Il-13 binding agents

ABSTRACT

Agents (e.g., antibodies and fragments thereof) that bind specifically to IL 13 and modulate the ability of IL-13 to interact with IL-13 receptors and signaling mediators are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/155,843, filed on Jun. 17, 2005. This application claims priority toU.S. Patent Application Ser. No. 60/581,078, filed on Jun. 17, 2004,under 35 U.S.C. §119, and is a continuation-in-part of U.S. patentapplication Ser. No. 11/149,025, filed on Jun. 9, 2005. The contents ofthe aforementioned applications are hereby incorporated by reference.This application also incorporates by reference PCT/US2005/021454, filedwith the U.S. Receiving Office on Jun. 17, 2005.

SEQUENCE LISTING

This application incorporates by reference the sequence listingsubmitted herewith in computer readable format (CRF) and paper format.

BACKGROUND

Interleukin-13 (IL-13) is a cytokine secreted by T lymphocytes and mastcells (McKenzie et al. (1993) Proc. Natl. Acad. Sci. USA 90:3735-39;Bost et al. (1996) Immunology 87:663-41). IL-13 shares severalbiological activities with IL-4. For example, either IL-4 or IL-13 cancause IgE isotype switching in B cells (Tomkinson et al. (2001) J.Immunol. 166:5792-5800). Additionally, increased levels of cell surfaceCD23 and serum CD23 (sCD23) have been reported in asthmatic patients(Sanchez-Guererro et al. (1994) Allergy 49:587-92; DiLorenzo et al.(1999) Allergy Asthma Proc. 20:119-25). In addition, either IL-4 orIL-13 can upregulate the expression of MHC class II and the low-affinityIgE receptor (CD23) on B cells and monocytes, which results in enhancedantigen presentation and regulated macrophage function (Tomkinson etal., supra). Importantly, either IL-4 or IL-13 can increase theexpression of VCAM-1 on endothelial cells, which facilitatespreferential recruitment of eosinophils (and T cells) to the airwaytissues (Tomkinson et al., supra). Either IL-4 or IL-13 can alsoincrease airway mucus secretion, which can exacerbate airwayresponsiveness (Tomkinson et al., supra). These observations suggestthat although IL-13 is not necessary for, or even capable of, inducingTh2 development, IL-13 may be a key player in the development of airwayeosinophilia and AHR (Tomkinson et al., supra; Wills-Karp et al. (1998)Science 282:2258-61).

SUMMARY

We have discovered, inter alia, IL-13 binding agents, in particular,anti-IL-13 antibody molecules can bind to human IL-13 and/or cynomolgusmonkey IL-13, with high affinity and specificity. In one embodiment, theantibody molecules reduce at least one IL-13-associated activity, e.g.,modulation of an inflammatory condition. For example, the anti-IL-13antibody molecules can bind to IL-13 and modulate, e.g., inhibit, aninteraction (e.g., binding) between IL-13 and an IL-13 receptor, e.g.,IL-13 receptor cd (“IL-13Rα1), IL-13 receptor a2 (“IL-13Rα2”), and/orthe interleukin-4 receptor alpha chain (“IL-4RI”), thereby reducing orpreventing signal transduction.

An IL-13 binding agent, such as an anti-IL-13 antibody molecule can beused to modulate (e.g., inhibit) at least one IL-13-associated activityin vivo. The IL-13 binding agent can be used to treat or prevent anIL-13 associated-disorder, or to ameliorate at least one symptomthereof. Exemplary IL-13 associated disorders include inflammatorydisorders (e.g., lung inflammation), respiratory disorders (e.g.,asthma, including allergic and non-allergic asthma, chronic obstructivepulmonary disease (COPD)), as well as conditions involving airwayinflammation, eosinophilia, fibrotic disorders (e.g., cystic fibrosis,liver fibrosis, and pulmonary fibrosis), scleroderma, excess mucusproduction; atopic disorders (e.g., atopic dermatitis, urticaria,eczema, allergic rhinitis, and allergic enterogastritis), an IL-13associated cancer (e.g., a leukemia, glioblastoma, or lymphoma, e.g.,Hodgkin's lymphoma), gastrointestinal disorders (e.g., inflammatorybowel diseases), liver disorders (e.g., cirrhosis), and viralinfections.

An IL-13 binding agent can be a protein, e.g., an antibody molecule, apeptide, or a scaffold domain, that interacts with, e.g., binds toand/or inhibits IL-13, in particular, mammalian IL-13, e.g., human ornonhuman primate IL-13. The antibody molecule can be an isolatedantibody molecule. In one embodiment, the binding agent is anantagonist, e.g., a binding agent that neutralizes, reduces and/orinhibits one or more IL-13-associated activities, including but notlimited to, induction of CD23 expression; production of IgE by human Bcells; phosphorylation of a transcription factor, e.g., STAT protein(e.g., STAT6 protein); antigen-induced eosinophilia in vivo;antigen-induced bronchoconstriction in vivo; or drug-induced airwayhyperreactivity in vivo, among others. For example, the binding agenthas a statistically significant effect in one or more assays describedherein. Beside anti-IL-13 antibody molecules, other IL-13 binding agentsthat can be used include IL-13 receptor-Fc fusions, other soluble formsof the IL-13 receptor, soluble forms of IL-4R1, antibodies that bind toIL-13R, and other molecules that inhibit the interaction between IL-13and one of its receptors.

In one aspect, the invention features an IL-13 binding agent that thatbinds to IL-13, e.g., with an affinity corresponding to a K_(D) of lessthan 5×10⁻⁷M, 1×10⁻⁷M, 5×10⁻⁸, 1×10⁻⁸, 5×10⁻⁹, 1×10⁻⁹ M, more typicallyless than 5×10⁻¹°, 1×10⁻¹⁰, 5×10⁻¹¹ M, 1×10⁻¹¹ M, or better. The IL-13binding agent can be, for example, an antibody molecule that includesfirst and second immunoglobulin variable domain sequences that includeat least a sufficient portion of an immunoglobulin variable domain toform an antigen-binding site that binds to IL-13. Typically, the firstand second immunoglobulin variable domain sequences correspond toimmunoglobulin variable domain sequences of a heavy and light chain,e.g., a paired or otherwise compatible heavy and light chain.

In one embodiment, the IL-13 binding agent binds to one or more of thefollowing peptides:

FVKDLLVHLKKLFREGQ₁₃₀FN (SEQ ID NO:1),

FVKDLLVHLKKLFREGR₁₃₀FN (SEQ ID NO:2),

FVKDLLLHLKKLFREGQ₁₃₀FN (SEQ ID NO:3),

FVKDLLLHLKKLFREGR₁₃₀FN (SEQ ID NO:4),

FVKDLLVHLKKLFREG (SEQ ID NO:5), and

FVKDLLLHLKKLFREG (SEQ ID NO:6), e.g., as isolated peptides, or to anamino acid within such a peptide when the peptide is folded in thestructure of a mature IL-13 protein.

For example, the IL-13 binding agent can bind to a peptide or to anIL-13 with comparable affinity (e.g., affinities that differ by lessthan a factor of 8, 5, 4, or 2), regardless of whether R or Q is presentat position 130. In particular, the IL-13 binding agent may bind withequal affinity to the peptide or the IL-13 regardless of whether R or Qis present at position 130.

The IL-13 binding agent may bind to one or more of the followingpeptides:

KDLLVHLKKLFREGQFN (SEQ ID NO:7),

KDLLVHLKKLFREGRFN (SEQ ID NO:8),

KDLLLHLKKLFREGQFN (SEQ ID NO:9),

KDLLLHLKKLFREGRFN (SEQ ID NO:10),

KDLLVHLKKLFRE (SEQ ID NO:11),

KDLLLHLKKLFRE (SEQ ID NO:12), and

HLKKLFRE (SEQ ID NO:13), e.g., as isolated peptides, or to an amino acidwithin such a peptide when the peptide is folded in the structure of amature IL-13 protein. The IL-13 binding agent can bind to an epitope onIL-13 that includes at least one (e.g., one, two, three or four) aminoacid residues from a peptide sequence recited herein (e.g., in FIG. 1B),or a corresponding peptide which differs by at least one, but no morethan one, two or three amino acid residues, e.g., a correspondingpeptide from human IL-13.

In one embodiment, the IL-13 binding agent contacts (e.g., makes a vander Waals contact with) an amino acid residue in helix D (amino acidresidues 114-130) of full-length IL-13 (SEQ ID NO:24 or SEQ ID NO:178),e.g., one or more of the following amino acid residues: residue 116,117, 118, 122, 123, 124, 125, 126, 127, or 128 of SEQ ID NO:24 or SEQ IDNO:178. In one embodiment, the IL-13 binding agent binds to an epitopeon helix D, or an epitope that includes at least one amino acid residue(e.g., at least one, two, three, or four) on helix D, and/or may inhibitinteraction of IL-13 with one or both of IL-13Rα1 and/or IL-13Rα2. HelixD corresponds to amino acid residues 95-111 of mature, processed IL-13(SEQ ID NO:14 or SEQ ID NO:124).

In one embodiment, the IL-13 binding agent specifically binds to anepitope, e.g., a linear or a conformational epitope, of IL-13, e.g.,mammalian, e.g., human IL-13. For example, the IL-13 binding agentcompetes with MJ 2-7 and/or C65 for binding to IL-13, e.g., to humanIL-13. The IL-13 binding agent may competitively inhibit binding of MJ2-7 and/or C65 to IL-13. The IL-13 binding agent may specifically bindat least one amino acid in an epitope defined by MJ 2-7 binding to humanIL-13 or an epitope defined by C65 binding to human IL-13. In oneembodiment, the IL-13 binding agent may bind to an epitope that overlapswith that of MJ 2-7 or C65, e.g., includes at least one, two, three, orfour amino acids in common, or an epitope that, when bound, stericallyprevents interaction with MJ 2-7 or C65.

In still another embodiment, the IL-13 binding agent specifically bindsat least one amino acid in an epitope defined by IL-13Rα1 binding tohuman IL-13 or an epitope defined by IL-13Rα2 binding to human IL-13, oran epitope that overlaps with such epitopes. The IL-13 binding agent maycompete with IL-13Rα1 and/or IL-13Rα2 for binding to IL-13, e.g., tohuman IL-13. The IL-13 binding agent may competitively inhibit bindingof IL-13Rα1 and/or IL-13Rα2 to IL-13. The IL-13 binding agent mayinteract with an epitope on IL-13 which, when bound, sterically preventsinteraction with IL-13Rα1 and/or IL-13Rα2.

In one embodiment, the IL-13 binding agent has a functional activitycomparable to IL-13Rα2, e.g., the IL-13 binding agent reduces orinhibits IL-13 interaction with IL-13Rα1. The IL-13 binding agent mayprevent formation of a complex between IL-13 and IL-13Rα1 or disrupt ordestabilize a complex between IL-13 and IL-13Rα1. In one embodiment, theIL-13 binding agent inhibits ternary complex formation, e.g., formationof a complex between IL 13, IL-13Rα1 and IL4-R.

In one embodiment, the IL-13 binding agent can inhibit one or moreIL-13-associated activities with an IC₅₀ of about 50 nM to 5 pM,typically about 100 to 250 pM or less, e.g., better inhibition. Agentsthat inhibit at least one activity of IL-13 are considered IL-13antagonists. In one embodiment, the IL-13 binding agent can associatewith IL-13 with kinetics in the range of 10³ to 10⁸ M⁻¹s⁻¹, typically10⁴ to 10⁷ M⁻¹s⁻¹. In yet another embodiment, the IL-13 binding agenthas dissociation kinetics in the range of 10⁻² to 10⁻⁶ s⁻¹, typically10⁻² to 10⁻⁵ s⁻¹. In one embodiment, the IL-13 binding agent binds toIL-13, e.g., human IL-13, with an affinity and/or kinetics similar(e.g., within a factor 20, 10, or 5) to monoclonal antibody MJ 2-7 orC65, or modified forms thereof, e.g., chimeric forms or humanized formsthereof (e.g., a humanized form described herein). The affinity andbinding kinetics of an IL-13 binding agent can be tested using, e.g.,biosensor technology (BIACORE™).

The IL-13 binding agent can be an antibody molecule, e.g., anantigen-binding fragment of an antibody (such as a Fab, F(ab′)2, Fv or asingle chain Fv fragment) or an antibody that includes an Fc domain.Typically, an anti-IL-13 antibody molecule is monoclonal or amono-specific.

The IL-13 binding agent, particularly an anti-IL-13 antibody molecule,can be an effectively human, human, humanized, CDR-grafted, chimeric,mutated, affinity matured, deimmunized, synthetic or otherwise invitro-generated protein. In one embodiment, the IL-13 binding agent is ahumanized antibody. In one embodiment, the IL-13 binding agent is notantigenic in humans or does not cause a HAMA response.

In one embodiment, the IL-13 antibody molecule includes a heavy andlight chain. The heavy and light chains of an anti-IL-13 antibodymolecule can be substantially full-length (e.g., an antibody moleculecan include at least one, and preferably two heavy chains, and at leastone, and preferably two light chains) or can include an antigen-bindingfragment (e.g., a Fab, F(ab′)2, Fv or a single chain Fv fragment). Inyet other embodiments, the antibody molecule has a heavy chain constantregion chosen from, e.g., the heavy chain constant regions of IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosenfrom, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, andIgG4, more particularly, the heavy chain constant regions IgG1 (e.g.,human IgG1). Typically the heavy chain constant region is human or amodified form of a human constant region (e.g., as described in Example5). In another embodiment, the antibody molecule has a light chainconstant region chosen from, e.g., the light chain constant regions ofkappa or lambda, preferably kappa (e.g., human kappa). In oneembodiment, the constant region is altered, e.g., mutated, to modify theproperties of the antibody molecule (e.g., to increase or decrease oneor more of: Fc receptor binding, antibody glycosylation, the number ofcysteine residues, effector cell function, or complement function). Forexample, the human IgG1 constant region can be mutated at one or moreresidues, e.g., one or more of residues 234 and 237, as described inExample 5.

In one embodiment, the IL-13 binding agent (e.g., the anti-IL-13 bindingmolecule) includes at least one, two and preferably three CDRs from thelight or heavy chain variable domain of an antibody disclosed herein,e.g., MJ 2-7. For example, the protein includes one or more of thefollowing sequences within a CDR region:

GFNIKDTYIH (SEQ ID NO:15),

RIDPANDNIKYDPKFQG (SEQ ID NO:16),

SEENWYDFFDY (SEQ ID NO:17),

RSSQSIVHSNGNTYLE (SEQ ID NO:18),

KVSNRFS (SEQ ID NO:19), and

FQGSHIPYT (SEQ ID NO:20), or a CDR having an amino acid sequence thatdiffers by no more than 4, 3, 2.5, 2, 1.5, 1, or 0.5 alterations (e.g.,substitutions, insertions or deletions) for every 10 amino acids (e.g.,the number of differences being proportional to the CDR length) relativeto a sequence listed above, e.g., at least one alteration but not morethan two, three, or four per CDR.

For example, the IL-13 binding agent can include, in the light chainvariable domain sequence, at least one, two, or three of the followingsequences within a CDR region:

RSSQSIVHSNGNTYLE (SEQ ID NO:18),

KVSNRFS (SEQ ID NO:19), and

FQGSHIPYT (SEQ ID NO:20), or an amino acid sequence that differs by nomore than 4, 3, 2.5, 2, 1.5, 1, or 0.5 substitutions, insertions ordeletions for every 10 amino acids relative to a sequence listed above.

The IL-13 binding agent can include, in the heavy chain variable domainsequence, at least one, two, or three of the following sequences withina CDR region:

GFNIKDTYIH (SEQ ID NO:15),

RIDPANDNIKYDPKFQG (SEQ ID NO:16), and

SEENWYDFFDY (SEQ ID NO:17), or an amino acid sequence that differs by nomore than 4, 3, 2.5, 2, 1.5, 1, or 0.5 substitutions, insertions ordeletions for every 10 amino acids relative to a sequence listed above.The heavy chain CDR3 region can be less than 13 or less than 12 aminoacids in length, e.g., 11 amino acids in length (either using Chothia orKabat definitions).

In another example, the IL-13 binding agent can include, in the lightchain variable domain sequence, at least one, two, or three of thefollowing sequences within a CDR region (amino acids in parenthesesrepresent alternatives for a particular position):

(i) (SEQ ID NO: 25) (RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS) or (SEQ ID NO: 26)(RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-E, or (SEQ ID NO: 21)(RK)-S-S-Q-S-(LI)-(KV)-H-S-N-G-N-T-Y-L-(EDNQYAS), (ii) (SEQ ID NO: 27)K-(LVI)-S-(NY)-(RW)-(FD)-S, or (SEQ ID NO: 22) K-(LV)-S-(NY)-R-F-S, and(iii) (SEQ ID NO: 28) Q-(GSA)-(ST)-(HEQ)-I-P, (SEQ ID NO: 23)F-Q-(GSA)-(SIT)-(HEQ)-(IL)-P, or (SEQ ID NO: 194)Q-(GSA)-(ST)-(HEQ)-I-P-Y-T, or (SEQ ID NO: 29)F-Q-(GSA)-(SIT)-(HEQ)-(IL)-P-Y-T.

In one preferred embodiment, the IL-13 binding agent includes all sixCDR's from MJ 2-7 or closely related CDRs, e.g., CDRs which areidentical or which have at least one amino acid alteration, but not morethan two, three or four alterations (e.g., substitutions, deletions, orinsertions). The IL-13 binding agent can include at least two, three,four, five, six, or seven IL-13 contacting amino acid residues of MJ 2-7

In still another example, the IL-13 binding agent includes at least one,two, or three CDR regions that have the same canonical structures andthe corresponding CDR regions of MJ 2-7, e.g., at least CDR1 and CDR2 ofthe heavy and/or light chain variable domains of MJ 2-7.

The IL-13 binding agent can include one of the following sequences:

(SEQ ID NO: 30) DIVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQG SHIPYT (SEQ ID NO: 31)DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQG SHIPYT (SEQ ID NO: 32)DIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQG SHIPYT (SEQ ID NO: 33)DIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQPPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQG SHIPYT (SEQ ID NO: 34)DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQG SHIPYT (SEQ ID NO: 35)DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWLQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQG SHIPYT (SEQ ID NO: 36)DIQMTQSPSSLSASVGDRVTITCRSSQSIVHSNGNTYLEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQG SHIPYT (SEQ ID NO: 37)DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQG SHIPYT (SEQ ID NO: 38)DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQG SHIPYTor a sequence that has fewer than eight, seven, six, five, four, three,or two alterations (e.g., substitutions, insertions or deletions, e.g.,conservative substitutions or a substitution for an amino acid residueat a corresponding position in MJ 2-7). Exemplary substitutions are atone of the following Kabat positions: 2, 4, 6, 35, 36, 38, 44, 47, 49,62, 64-69, 85, 87, 98, 99, 101, and 102. The substitutions can, forexample, substitute an amino acid at a corresponding position from MJ2-7 into a human framework region.

The IL-13 binding agent may also include one of the following sequences:

(SEQ ID NO: 39) DIVMTQTPLSLPVTPGEPASISC-(RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WYLQKPGQSPQLLIYK- (LVI)-S-(NY)-(RW)-(FD)-SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC F-Q-(GSA)- (SIT)-(HEQ)-(IL)-P(SEQ ID NO: 40) DVVMTQSPLSLPVTLGQPASISC-(RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WFQQRPGQSPRRLIYK- (LVI)-S-(NY)-(RW)-(FD)-SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF-Q-(GSA)-(SIT)- (HEQ)-(IL)-P(SEQ ID NO: 41) DIVMTQTPLSLSVTPGQPASISC-(RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WYLQKPGQSPQLLIYK- (LVI)-S-(NY)-(RW)-(FD)-SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF-Q-(GSA)-(SIT)- (HEQ)-(IL)-P(SEQ ID NO: 42) DIVMTQTPLSLSVTPGQPASISC(RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WYLQKPGQPPQLLIYK- (LVI)-S-(NY)-(RW)-(FD)-SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF-Q-(GSA)-(SIT)- (HEQ)-(IL)-P(SEQ ID NO: 43) DIVMTQSPLSLPVTPGEPASISC(RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WYLQKPGQSPQLLIYK- (LVI)-S-(NY)-(RW)-(FD)-SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF-Q-(GSA)-(SIT)- (HEQ)-(IL)-P(SEQ ID NO: 44) DIVMTQTPLSSPVTLGQPASISC(RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WLQQRPGQPPRLLIYK- (LVI)-S-(NY)-(RW)-(FD)-SGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCF-Q-(GSA)-(SIT)- (HEQ)-(IL)-P(SEQ ID NO: 45) DIQMTQSPSSLSASVGDRVTITC(RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WYQQKPGKAPKLLIYK- (LVI)-S-(NY)-(RW)-(FD)-SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCF-Q-(GSA)-(SIT)- (HEQ)-(IL)-P(SEQ ID NO: 46) DVLMTQTPLSLPVSLGDQASISC(RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WYLQKPGQSPKLLIYK- (LVI)-S-(NY)-(RW)-(FD)-SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCF-Q-(GSA)-(SIT)- (HEQ)-(IL)-Por a sequence that has fewer than eight, seven, six, five, four, three,or two alterations (e.g., substitutions, insertions or deletions, e.g.,conservative substitutions or a substitution for an amino acid residueat a corresponding position in MJ 2-7) in the framework region.Exemplary substitutions are at one or more of the following Kabatpositions: 2, 4, 6, 35, 36, 38, 44, 47, 49, 62, 64-69, 85, 87, 98, 99,101, and 102. The substitutions can, for example, substitute an aminoacid at a corresponding position from MJ 2-7 into a human frameworkregion. The sequences may also be followed by the dipeptide Tyr-Thr. TheFR4 region can include, e.g., the sequence FGGGTKVEIKR (SEQ ID NO:47).

In another example, the IL-13 binding agent can include, in the heavychain variable domain sequence, at least one, two, or three of thefollowing sequences within a CDR region (amino acids in parenthesesrepresent alternatives for a particular position):

(i) (SEQ ID NO: 48) G-(YF)-(NT)-I-K-D-T-Y-(MI)-H, (ii) (SEQ ID NO: 49)(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-G, and (iii) (SEQ ID NO: 17)SEENWYDFFDY.

The IL-13 binding agent can include one of the following sequences:

(SEQ ID NO: 50) QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGRIDPANDNIKYDPKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 51) QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQRLEWMGRIDPANDNIKYDPKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 52) QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQATGQGLEWMGRIDPANDNIKYDPKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 53) QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGRIDPANDNIKYDPKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 54) QVQLVQSGAEVKKPGASVKVSCKVSGFNIKDTYIHWVRQAPGKGLEWMGRIDPANDNIKYDPKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT SEENWYDFFDY(SEQ ID NO: 55) QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDTYIHWVRQAPGQALEWMGRIDPANDNIKYDPKFQGRVTITRDRSMSTAYMELSSLRSEDTAMYYCAR SEENWYDFFDY(SEQ ID NO: 56) QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGRIDPANDNIKYDPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 57) QMQLVQSGPEVKKPGTSVKVSCKASGFNIKDTYIHWVRQARGQRLEWIGRIDPANDNIKYDPKFQGRVTITRDMSTSTAYMELSSLRSEDTAVYYCAA SEENWYDFFDY(SEQ ID NO: 58) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 59) EVQLVESGGGLVQPGRSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVSRIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAK DSEENWYDFFDY(SEQ ID NO: 60) QVQLVESGGGLVKPGGSLRLSCAASGFNIKDTYIHWIRQAPGKGLEWVSRIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 61) EVQLVESGGGLVKPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVGRIDPANDNIKYDPKFQGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT SEENWYDFFDY(SEQ ID NO: 62) EVQLVESGGGVVRPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVSRIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTALYHCAR SEENWYDFFDY(SEQ ID NO: 63) EVQLVESGGGLVKPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVSRIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 64) EVQLLESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVSRIDPANDNIKYDPKFQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SEENWYDFFDY(SEQ ID NO: 65) QVQLVESGGGVVQPGRSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIDPANDNIKYDPKFQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SEENWYDFFDY(SEQ ID NO: 66) QVQLVESGGGVVQPGRSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIDPANDNIKYDPKFQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 67) EVQLVESGGVVVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVSRIDPANDNIKYDPKFQGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAK DSEENWYDFFDY(SEQ ID NO: 68) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVSRIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 69) EVQLVESGGGLVQPGRSLRLSCTASGFNIKDTYIHWFRQAPGKGLEWVGRIDPANDNIKYDPKFQGRFTISRDGSKSIAYLQMNSLKTEDTAVYYCTR SEENWYDFFDY(SEQ ID NO: 70) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEYVSRIDPANDNIKYDPKFQGRFTISRDNSKNTLYLQMGSLRAEDMAVYYCAR SEENWYDFFDY(SEQ ID NO: 71) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWIGRIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 72) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIDPANDNIKYDPKFQGKATISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 73) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIDPANDNIKYDPKFQGRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 74) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVGRIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 75) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIDPANDNIKYDPKFQGKATISADNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 76) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWIGRIDPANDNIKYDPKFQGRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 77) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVGRIDPANDNIKYDPKFQGRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 78) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIDPANDNIKYDPKFQGRFTISRDNAKNSAYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 79) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVGRIDPANDNIKYDPKFQGRFTISADNAKNSAYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 80) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWIGRIDPANDNIKYDPKFQGRFTISADNAKNSAYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 81) EVQLVESGGGLVQPGGSLRLSCTGSGFNIKDTYIHWVRQAPGKGLEWIGRIDPANDNIKYDPKFQGRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 82) EVQLQQSGAELVKPGASVKLSCTGSGFNIKDTYIHWVKQRPEQGLEWIGRIDPANDNIKYDPKFQGKATITADTSSNTAYLQLNSLTSEDTAVYYCAR SEENWYDFFDYor a sequence that has fewer than eight, seven, six, five, four, three,or two alterations (e.g., substitutions, insertions or deletions, e.g.,conservative substitutions or a substitution for an amino acid residueat a corresponding position in MJ 2-7). Exemplary substitutions are atone or more of the following Kabat positions: 2, 4, 6, 25, 36, 37, 39,47, 48, 93, 94, 103, 104, 106, and 107. Exemplary substitutions can alsobe at one or more of the following positions (accordingly to sequentialnumbering): 48, 49, 67, 68, 72, and 79. The substitutions can, forexample, substitute an amino acid at a corresponding position from MJ2-7 into a human framework region. In one embodiment, the sequenceincludes (accordingly to sequential numbering) one or more of thefollowing: Ile at 48, Gly at 49, Lys at 67, Ala at 68, Ala at 72, andAla at 79; preferably, e.g., Be at 48, Gly at 49, Ala at 72, and Ala at79.

Further, the frameworks of the heavy chain variable domain sequence caninclude: (i) at a position corresponding to 49, Gly; (ii) at a positioncorresponding to 72, Ala; (iii) at positions corresponding to 48, Ile,and to 49, Gly; (iv) at positions corresponding to 48, Ile, to 49, Gly,and to 72, Ala; (v) at positions corresponding to 67, Lys, to 68, Ala,and to 72, Ala; and/or (vi) at positions corresponding to 48, Ile, to49, Gly, to 72, Ala, to 79, Ala.

The IL-13 binding agent may also include one of the following sequences:

(SEQ ID NO: 83) QVQLVQSGAEVKKPGASVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGQGLEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 84) QVQLVQSGAEVKKPGASVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGQRLEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRVTITRDTSASTAYMELSSLRSEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 85) QVQLVQSGAEVKKPGASVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQATGQGLEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRVTMTRNTSISTAYMELSSLRSEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 86) QVQLVQSGAEVKKPGASVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGQGLEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 87) QVQLVQSGAEVKKPGASVKVSCKVSG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT SEENWYDFFDY(SEQ ID NO: 88) QMQLVQSGAEVKKTGSSVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGQALEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRVTITRDRSMSTAYMELSSLRSEDTAMYYCAR SEENWYDFFDY(SEQ ID NO: 89) QVQLVQSGAEVKKPGASVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGQGLEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 90) QMQLVQSGPEVKKPGTSVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQARGQRLEWIG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRVTITRDMSTSTAYMELSSLRSEDTAVYYCAA SEENWYDFFDY(SEQ ID NO: 91) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 92) EVQLVESGGGLVQPGRSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTALYYCAK DSEENWYDFFDY(SEQ ID NO: 93) QVQLVESGGGLVKPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WIRQAPGKGLEWVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 94) EVQLVESGGGLVKPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT SEENWYDFFDY(SEQ ID NO: 95) EVQLVESGGGVVRPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTALYHCAR SEENWYDFFDY(SEQ ID NO: 96) EVQLVESGGGLVKPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 97) EVQLLESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SEENWYDFFDY(SEQ ID NO: 98) QVQLVESGGGVVQPGRSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK SEENWYDFFDY(SEQ ID NO: 99) QVQLVESGGGVVQPGRSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 100) EVQLVESGGVVVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNSKNSLYLQMNSLRTEDTALYYCAK DSEENWYDFFDY(SEQ ID NO: 101) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 102) EVQLVESGGGLVQPGRSLRLSCTASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WFRQAPGKGLEWVG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDGSKSIAYLQMNSLKTEDTAVYYCTR SEENWYDFFDY(SEQ ID NO: 103) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEYVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNSKNTLYLQMGSLRAEDMAVYYCAR SEENWYDFFDY(SEQ ID NO: 104) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWIG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 105) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GKATISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 106) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 107) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 108) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GKATISADNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 109) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWIG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 110) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 111) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSAYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 112) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWVG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISADNAKNSAYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 113) EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWIG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISADNAKNSAYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 114) EVQLVESGGGLVQPGGSLRLSCTGSG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVRQAPGKGLEWIG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR SEENWYDFFDY(SEQ ID NO: 115) EVQLQQSGAELVKPGASVKLSCTGSG-(YF)-(NT)-I-K-D-T-Y-(MI)-H,WVKQRPEQGLEWIG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-GKATITADTSSNTAYLQLNSLTSEDTAVYYCAR SEENWYDFFDYor a sequence that has fewer than eight, seven, six, five, four, three,or two alterations (e.g., substitutions, insertions or deletions, e.g.,conservative substitutions or a substitution for an amino acid residueat a corresponding position in MJ 2-7) in the framework region.Exemplary substitutions are at one or more of the following Kabatpositions: 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106, and107. The substitutions can, for example, substitute an amino acid at acorresponding position from MJ 2-7 into a human framework region. TheFR4 region can include, e.g., the sequence WGQGTTLTVSS (SEQ ID NO:116)or WGQGTLVTVSS (SEQ ID NO:117).

In one embodiment, the heavy chain variable domain sequence is at least90, 92, 93, 94, 95, 96, 97, 98, 99% identical or identical to the heavychain variable domain of V2.1, V2.2, V2.3, V2.4, V2.5, V2.6, V2.7,V2.11, or other heavy chain variable domain described herein. In oneembodiment, the heavy chain variable domain sequence includes variabledomain sequence comprises a sequence encoded by a nucleic acid thathybridizes under high stringency conditions to the complement of anucleic acid encoding the heavy chain variable domain of V2.1, V2.2,V2.3, V2.4, V2.5, V2.6, V2.7, V2.11, or other heavy chain variabledomain described herein. In one embodiment, the light chain variabledomain sequence is at least 90, 92, 93, 94, 95, 96, 97, 98, 99%identical or identical to the light chain variable domain of V2.11 orother light chain variable domain described herein. In one embodiment,the light chain variable domain sequence comprises a sequence encoded bya nucleic acid that hybridizes under high stringency conditions to thecomplement of a nucleic acid encoding the light chain variable domain ofV2.11 or other light chain variable domain described herein.

In one embodiment, the heavy chain framework (e.g., FR1, FR2, FR3,individually, or a sequence encompassing FR1, FR2, and FR3, butexcluding CDRs) includes an amino acid sequence, which is at least 80%,85%, 90%, 95%, 97%, 98%, 99% or higher identical to the heavy chainframework of one of the following germline V segment sequences: DP-25,DP-1, DP-12, DP-9, DP-7, DP-31, DP-32, DP-33, DP-58, or DP-54, oranother V gene which is compatible with the canonical structure class1-3 (see, e.g., Chothia et al. (1992) J. Mol. Biol. 227:799-817;Tomlinson et al. (1992) J. Mol. Biol. 227:776-798). Other frameworkscompatible with the canonical structure class 1-3 include frameworkswith the one or more of the following residues according to Kabatnumbering: Ala, Gly, Thr, or Val at position 26; Gly at position 26;Tyr, Phe, or Gly at position 27; Phe, Val, Ile, or Leu at position 29;Met, Ile, Leu, Val, Thr, Trp, or Ile at position 34; Arg, Thr, Ala, Lysat position 94; Gly, Ser, Asn, or Asp at position 54; and Arg atposition 71.

In one embodiment, the light chain framework (e.g., FR1, FR2, FR3,individually, or a sequence encompassing FR1, FR2, and FR3, butexcluding CDRs) includes an amino acid sequence, which is at least 80%,85%, 90%, 95%, 97%, 98%, 99% or higher identical to the light chainframework of a Vκ II subgroup germline sequence or one of the followinggermline V segment sequences: A17, A1, A18, A2, A19/A3, or A23 oranother V gene which is compatible with the canonical structure class4-1 (see, e.g., Tomlinson et al. (1995) EMBO J. 14:4628). Otherframeworks compatible with the canonical structure class 4-1 includeframeworks with the one or more of the following residues according toKabat numbering: Val or Leu or Be at position 2; Ser or Pro at position25; Ile or Leu at position 29; Gly at position 31d; Phe or Leu atposition 33; and Phe at position 71.

In another embodiment, the light chain framework (e.g., FR1, FR2, FR3,individually, or a sequence encompassing FR1, FR2, and FR3, butexcluding CDRs) includes an amino acid sequence, which is at least 80%,85%, 90%, 95%, 97%, 98%, 99% or higher identical to the light chainframework of a Vκ I subgroup germline sequence, e.g., a DPK9 sequence.

In another embodiment, the heavy chain framework (e.g., FR1, FR2, FR3,individually, or a sequence encompassing FR1, FR2, and FR3, butexcluding CDRs) includes an amino acid sequence, which is at least 80%,85%, 90%, 95%, 97%, 98%, 99% or higher identical to the light chainframework of a VH I subgroup germline sequence, e.g., a DP-25 sequenceor a VH III subgroup germline sequence, e.g., a DP-54 sequence.

In one embodiment, the IL-13 binding agent includes at least one, twoand preferably three CDR's from the light or heavy chain variable domainof an antibody disclosed herein, e.g., C65. For example, the IL-13binding agent includes one or more of the following sequences within aCDR region:

QASQGTSINLN (SEQ ID NO:118),

GASNLED (SEQ ID NO:119), and

LQHSYLPWT (SEQ ID NO:120)

GFSLTGYGVN (SEQ ID NO:121),

IIWGDGSTDYNSAL (SEQ ID NO:122), and

DKTFYYDGFYRGRMDY (SEQ ID NO:123), or a CDR having an amino acid sequencethat differs by no more than 4, 3, 2.5, 2, 1.5, 1, or 0.5 substitutions,insertions or deletions for every 10 amino acids (e.g., the number ofdifferences being proportional to the CDR length) relative to a sequencelisted above, e.g., at least one alteration but not more than two,three, or four per CDR. For example, the protein can include, in thelight chain variable domain sequence, at least one, two, or three of thefollowing sequences within a CDR region:

QASQGTSINLN (SEQ ID NO:118),

GASNLED (SEQ ID NO:119), and

LQHSYLPWT (SEQ ID NO:120), or an amino acid sequence that differs by nomore than 4, 3, 2.5, 2, 1.5, 1, or 0.5 substitutions, insertions ordeletions for every 10 amino acids relative to a sequence listed above.

The IL-13 binding agent can include, in the heavy chain variable domainsequence, at least one, two, or three of the following sequences withina CDR region:

GFSLTGYGVN (SEQ ID NO:121),

IIWGDGSTDYNSAL (SEQ ID NO:122), and

DKTFYYDGFYRGRMDY (SEQ ID NO:123), or an amino acid sequence that differsby no more than 4, 3, 2.5, 2, 1.5, 1, or 0.5 substitutions, insertionsor deletions for every 10 amino acids relative to a sequence listedabove.

In one preferred embodiment, the IL-13 binding agent includes all sixCDRs from C65 or closely related CDRs, e.g., CDRs which are identical orwhich have at least one amino acid alteration, but not more than two,three or four alterations (e.g., substitutions, deletions, orinsertions).

In still another embodiment, the IL-13 binding agent includes at leastone, two or three CDR regions that have the same canonical structuresand the corresponding CDR regions of C65, e.g., at least CDR1 and CDR2of the heavy and/or light chain variable domains of C65.

In one embodiment, the heavy chain framework (e.g., FR1, FR2, FR3,individually, or a sequence encompassing FR1, FR2, and FR3, butexcluding CDRs) includes an amino acid sequence, which is at least 80%,85%, 90%, 95%, 97%, 98%, 99% or higher identical to the heavy chainframework of one of the following germline V segment sequences: DP-71 orDP-67 or another V gene which is compatible with the canonical structureclass of C65 (see, e.g., Chothia et al. (1992) J. Mol. Biol.227:799-817; Tomlinson et al. (1992) J. Mol. Biol. 227:776-798).

In one embodiment, the light chain framework (e.g., FR1, FR2, FR3,individually, or a sequence encompassing FR1, FR2, and FR3, butexcluding CDRs) includes an amino acid sequence, which is at least 80%,85%, 90%, 95%, 97%, 98%, 99% or higher identical to the light chainframework of DPK-1 or DPK-9 germline sequence or another V gene which iscompatible with the canonical structure class of C65 (see, e.g.,Tomlinson et al. (1995) EMBO J. 14:4628).

In another embodiment, the light chain framework (e.g., FR1, FR2, FR3,individually, or a sequence encompassing FR1, FR2, and FR3, butexcluding CDRs) includes an amino acid sequence, which is at least 80%,85%, 90%, 95%, 97%, 98%, 99% or higher identical to the light chainframework of a Vκ I subgroup germline sequence, e.g., a DPK-9 or DPK-1sequence.

In another embodiment, the heavy chain framework (e.g., FR1, FR2, FR3,individually, or a sequence encompassing FR1, FR2, and FR3, butexcluding CDRs) includes an amino acid sequence, which is at least 80%,85%, 90%, 95%, 97%, 98%, 99% or higher identical to the light chainframework of a VH IV subgroup germline sequence, e.g., a DP-71 or DP-67sequence.

In one embodiment, the light or the heavy chain variable framework(e.g., the region encompassing at least FR1, FR2, FR3, and optionallyFR4) can be chosen from: (a) a light or heavy chain variable frameworkincluding at least 80%, 85%, 90%, 95%, or 100% of the amino acidresidues from a human light or heavy chain variable framework, e.g., alight or heavy chain variable framework residue from a human matureantibody, a human germline sequence, a human consensus sequence, or ahuman antibody described herein; (b) a light or heavy chain variableframework including from 20% to 80%, 40% to 60%, 60% to 90%, or 70% to95% of the amino acid residues from a human light or heavy chainvariable framework, e.g., a light or heavy chain variable frameworkresidue from a human mature antibody, a human germline sequence, a humanconsensus sequence; (c) a non-human framework (e.g., a rodentframework); or (d) a non-human framework that has been modified, e.g.,to remove antigenic or cytotoxic determinants, e.g., deimmunized, orpartially humanized. In one embodiment, the heavy chain variable domainsequence includes human residues or human consensus sequence residues atone or more of the following positions (preferably at least five, ten,twelve, or all): (in the FR of the variable domain of the light chain)4L, 35L, 36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L, 65L, 66L, 67L,68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, and/or (in the FR of thevariable domain of the heavy chain) 2H, 4H, 24H, 36H, 37H, 39H, 43H,45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H, 75H, 78H, 91H, 92H,93H, and/or 103H (according to the Kabat numbering).

In one embodiment, the IL-13 binding agent includes at least onenon-human CDR, e.g., a murine CDR, e.g., a CDR from MJ 2-7 or C65, or amutant thereof, and at least one framework which differs from aframework of MJ 2-7 or C65 by at least one amino acid, e.g., at least 5,8, 10, 12, 15, or 18 amino acids. For example, the proteins include one,two, three, four, five, or six such non-human CDRs and includes at leastone amino acid difference in at least three of HC FR1, HC FR2, HC FR3,LC FR1, LC FR2, and LC FR3.

In one embodiment, the heavy or light chain variable domain sequence ofthe anti-IL-13 antibody molecule includes an amino acid sequence, whichis at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical to avariable domain sequence of an antibody described herein, e.g., MJ 2-7or C65; or which differs at least 1 or 5 residues, but less than 40, 30,20, or 10 residues, from a variable domain sequence of an antibodydescribed herein, e.g., MJ 2-7 or C65. In one embodiment, the heavy orlight chain variable domain sequence of the protein includes an aminoacid sequence encoded by a nucleic acid sequence described herein or anucleic acid that hybridizes to a nucleic acid sequence described hereinor its complement, e.g., under low stringency, medium stringency, highstringency, or very high stringency conditions.

In one embodiment, one or both of the variable domain sequences includeamino acid positions in the framework region that are variously derivedfrom both a non-human antibody (e.g., a murine antibody such as mAb13.2)and a human antibody or germline sequence. For example, a variabledomain sequence can include a number of positions at which the aminoacid residue is identical to both the non-human antibody and the humanantibody (or human germline sequence) because the two are identical atthat position. Of the remaining framework positions where the non-humanand human differ, at least 50, 60, 70, 80, or 90% of the positions ofthe variable domain are preferably identical to the human antibody (orhuman germline sequence) rather than the non-human. For example, none,or at least one, two, three, or four of such remaining frameworkposition may be identical to the non-human antibody rather than to thehuman. For example, in HC FR1, one or two such positions can benon-human; in HC FR2, one or two such positions can be non-human; inFR3, one, two, three, or four such positions can be non-human; in LCFR1, one, two, three, or four such positions can be non-human; in LCFR2, one or two such positions can be non-human; in LC FR3, one or twosuch positions can be non-human. The frameworks can include additionalnon-human positions.

The IL-13 binding agent, e.g., anti-IL-13 antibody molecule, can bederivatized or linked to another functional molecule, e.g., anotherpeptide or protein (e.g., an Fab fragment). For example, the bindingagent can be functionally linked (e.g., by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody molecule (e.g., to form abispecific or a multispecific antibody molecule), toxins, radioisotopes,cytotoxic or cytostatic agents, among others.

In another embodiment, the IL-13 binding agent, e.g., anti-IL-13antibody molecule, interferes with the interaction of IL-13 with thereceptor IL-13RI1. In one embodiment, the IL-13 binding agent caninterfere with the interaction of Phe107 of IL-13 (SEQ ID NO:124; FIG.13A) with a hydrophobic pocket of IL-13Rα1 formed by the side chains ofresidues Leu319, Cys257, Arg256, and Cys320 (SEQ ID NO:125; FIG. 13B),e.g., by direct binding to these residues or steric hindrance. Inanother embodiment, the IL-13 binding agent can interfere with van derWaals interactions between amino acid residues Ile254, Ser255, Arg256,Lys318, Cys320, and Tyr321 of IL-13Rα1 (SEQ ID NO:125) and amino acidresidues Arg11, Glu12, Leu13, Ile14, Glu15, Lys104, Lys105, Leu106,Phe107, and Arg108 of IL-13 (SEQ ID NO:124), e.g., by direct binding tothese residues or steric hindrance.

In one embodiment, the IL-13 binding agent, e.g., the anti-IL-13antibody, molecule has no significant cross-reactivity when screenedagainst at least half, two-thirds, three-quarter, 90%, or all thetissues on the “suggested list of human tissues to be used forimmunohistochemical investigations of cross-reactivity” in Annex II ofthe DC CPMP Guideline III/5271/94 Draft 5, “Production and qualitycontrol of monoclonal antibodies” and at least half, two-thirds,three-quarter, 90%, or all of the tissues recommended in Table 2 of the1997 US FDA/CBER “Points to Consider in the Manufacture and Testing ofMonoclonal Antibody Products for Human Use.”

In one embodiment, the IL-13 binding agent, e.g., the anti-IL-13antibody, specifically binds to IL-13, e.g., a mammalian IL-13, e.g.,human or non-human primate IL-13. For example, the binding agent bindsto IL-13 with an affinity that is at least two-fold, 10-fold, 50-fold,100-fold, or better (smaller K_(d)) than its affinity for binding to anon-specific antigen (e.g., BSA, casein) other than IL-13, or with anaffinity that is at least two-fold, 10-fold, 50-fold, 100-fold, orbetter (smaller K_(d)) than its affinity for binding to another humaninterleukin other than IL-13. In some embodiments, the IL-13 bindingagent only detects a single prominent band when blotted against thecrude sample of human IL-13 described in Example 1 (“QuaternaryScreen”). In some embodiments, a precipitate made by pulling downproteins from that crude sample using beads to which the IL-13 bindingagent is immobilized is a composition in which IL-13 is at least 5%,10%, 50%, or 80% pure.

In another aspect, an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, or a pharmaceutical composition thereof, is administered totreat or prevent an IL-13-associated disorder. Treating refers toimproving or maintaining (or so attempting) the condition of subject. Ina typical case, treating improves the condition of the subject to anextent discernable to a physician or prevents worsening of thecondition. Examples of IL-13-associated disorders include, but are notlimited to, disorders chosen from one or more of: respiratory disorders,e.g., asthma (e.g., allergic and nonallergic asthma (e.g., asthma due toinfection with, e.g., respiratory syncytial virus (RSV), e.g., inyounger children)), chronic obstructive pulmonary disease (COPD), andother conditions involving airway inflammation, eosinophilia, fibrosisand excess mucus production, e.g., cystic fibrosis and pulmonaryfibrosis; atopic disorders, e.g., resulting from an increasedsensitivity to IL-13, (e.g., atopic dermatitis, urticaria, eczema,allergic rhinitis, and allergic enterogastritis); inflammatory and/orautoimmune conditions of, the skin (e.g., atopic dermatitis),gastrointestinal disorders (e.g., inflammatory bowel diseases (IBD),such as ulcerative colitis and/or Crohn's disease), liver (e.g.,cirrhosis, hepatocellular carcinoma), and scleroderma; tumors or cancers(e.g., soft tissue or solid tumors), such as leukemia, glioblastoma, andlymphoma, e.g., Hodgkin's lymphoma; viral infections (e.g., fromHTLV-1); fibrosis of other organs, e.g., fibrosis of the liver, (e.g.,fibrosis caused by a hepatitis B and/or C virus); and suppression ofexpression of protective type 1 immune responses, (e.g., duringvaccination), e.g., as described herein.

The IL-13 binding agent (e.g., the anti-IL-13 antibody molecule, such asone described herein) can be in administered in an amount effective totreat or prevent the disorder. In the case of prophylactic use (e.g., toprevent onset or delay onset), the subject may or may not have one ormore symptoms of the disorder. The amount can also be selected to beeffective to ameliorate at least one symptom of the disorder.Preferably, the subject is a mammal, e.g., a human suffering from anIL-13-associated disorder as described herein. For respiratorydisorders, e.g., asthma, the IL-13 binding agent can be delivered byinhalation.

In one embodiment, the method includes administering doses of anantibody molecule that binds to IL-13. For example, the antibodymolecule inhibits or neutralizes IL-13. In one embodiment, each dose isadministered subcutaneously, e.g., in an amount of about 0.5-10 mg/kg(e.g., 0.7-3.3 mg/kg) at a frequency of no more than once per week,e.g., every other week or once or twice monthly. In one embodiment, theantibody is an antibody described herein. For example, the antibody isan antibody that inhibits binding of IL-13Rα1. The antibody can, e.g.,confers a post-injection protective effect against exposure to Ascarisantigen in a sheep model at least 6 weeks after injection.

In one embodiment, the IL-13 binding agent is administered incombination with another therapeutic agent. The combination therapy caninclude an IL-13 binding agent, e.g., an anti-IL-13 antibody molecule,coformulated with and/or coadministered with one or more additionaltherapeutic agents, e.g., one or more cytokine and growth factorinhibitors, immunosuppressants, anti-inflammatory agents (e.g., systemicanti-inflammatory agents), metabolic inhibitors, enzyme inhibitors,and/or cytotoxic or cytostatic agents, as described in more herein. TheIL-13 binding agent and the other therapeutic can also be administeredseparately.

Examples of preferred additional therapeutic agents that can becoadministered and/or coformulated with an IL-13 binding agent include:inhaled steroids; beta-agonists, e.g., short-acting or long-actingbeta-agonists; antagonists of leukotrienes or leukotriene receptors;combination drugs such as ADVAIR®; IgE inhibitors, e.g., anti-IgEantibodies (e.g., XOLAIR®); phosphodiesterase inhibitors (e.g., PDE4inhibitors); xanthines; anticholinergic drugs; mast cell-stabilizingagents such as cromolyn; IL-4 inhibitors; IL-5 inhibitors; eotaxin/CCR3inhibitors; and antihistamines. Such combinations can be used to treatasthma and other respiratory disorders. Additional examples oftherapeutic agents that can be coadministered and/or coformulated withan IL-13 binding agent include one or more of: TNF antagonists (e.g., asoluble fragment of a TNF receptor, e.g., p55 or p75 human TNF receptoror derivatives thereof, e.g., 75 kd TNFR-IgG (75 kD TNF receptor-IgGfusion protein, ENBREL™)); TNF enzyme antagonists, e.g., TNFα convertingenzyme (TACE) inhibitors; muscarinic receptor antagonists; TGF-υantagonists; interferon gamma; perfenidone; chemotherapeutic agents,e.g., methotrexate, leflunomide, or a sirolimus (rapamycin) or an analogthereof, e.g., CCI-779; COX2 and cPLA2 inhibitors; NSAIDs;immunomodulators; p38 inhibitors, TPL-2, Mk-2 and NFPB inhibitors, amongothers.

In another aspect, this application provides compositions, e.g.,pharmaceutical compositions, that include a pharmaceutically acceptablecarrier and at least one IL-13 binding agent, e.g., an anti-IL-13antibody molecule. In one embodiment, the compositions, e.g.,pharmaceutical compositions, comprise a combination of two or more IL-13binding agents, e.g., two or more anti-IL-13 antibody molecules.Combinations of the IL-13 binding agent, e.g., the anti-IL-13 antibodymolecule, and a drug, e.g., a therapeutic agent (e.g., one or morecytokine and growth factor inhibitors, immunosuppressants,anti-inflammatory agents (e.g., systemic anti-inflammatory agents),metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostaticagents, as described herein, can also be used.

This application also features nucleic acids that include nucleotidesequences that encode an IL-13 binding agent described herein or acomponent thereof, e.g., a heavy and/or light chain variable domainsequence of an anti-IL-13 antibody molecule, e.g., an antibody moleculedescribed herein. For example, the application features a first andsecond nucleic acid encoding heavy and light chain variable domainsequences, respectively, of an anti-IL-13 antibody chosen from one ormore of, e.g., MJ 2-7 or C65, e.g., as described herein. In anotheraspect, the application features host cells and vectors containing thenucleic acids described herein.

The invention also features the epitope of IL-13, e.g., human IL-13,recognized by one or more of, e.g., MJ 2-7 or C65. For example, proteinsand peptides that include the epitope can be used to generate or screenfor other binding compounds that interact with the epitope, e.g.,proteins such as antibodies or small molecules. For example, a peptidethat includes the epitope can be used as an immunogen or as a target forscreening an expression library. It is also possible to evaluatecompounds for ability to interact with the peptide, or, by mapping orstructure determination, to evaluate compounds for ability to interactwith the epitope, e.g., in the context of a mature IL-13.

In another aspect, this application features a method of modulating,e.g., interfering with (e.g., inhibiting, blocking or otherwisereducing), an interaction, e.g., binding, between IL-13 and a cognateIL-13 binding protein, e.g., an IL-13 receptor complex, e.g., a complexcomprising IL-13RI1 and IL-4R1, or a subunit thereof. The modulating canbe effected in vivo or in vitro. In other embodiments, the IL-13 bindingagent, e.g., the anti-IL-13 antibody molecule, binds to IL-13, andinterferes with (e.g., inhibits, blocks or otherwise reduces) aninteraction, e.g., binding, between IL-13 and a subunit of the IL-13receptor complex, e.g., IL-13RI1 or IL-4R1, individually. In yet anotherembodiment, the IL-13 binding agent, e.g., the anti-IL-13 antibodymolecule, binds to IL-13, and interferes with (e.g., inhibits, blocks orotherwise reduces) an interaction, e.g., binding, between IL-13 andIL-13RI1. In another embodiment, the IL-13 binding agent, e.g., theanti-IL-13 antibody molecule, binds to IL-13, and interferes with (e.g.,inhibits, blocks or otherwise reduces) an interaction, e.g., binding,between IL-13 and IL-13RI1. Typically, the anti-IL-13 antibody moleculeinterferes with (e.g., inhibits, blocks or otherwise reduces) aninteraction, e.g., binding, of IL-13 and IL-13RI1.

In another aspect, this application features a method of modulatinginteraction between IL-13 and an IL-13 receptor protein, e.g., IL-13Rα1or IL-13Rα2. For example, an IL-13 binding agent, e.g., an agentdescribed herein, can be used to reduce or inhibit binding, betweenIL-13 and IL-13Rα1 or IL-13Rα2, or to reduce formation of a complex thatincludes IL-13Rα1 and IL-4RI (e.g., a complex as described herein). Themethod comprises contacting IL-13 or a complex that contains IL-13 withan IL-13 binding agent, e.g., a protein described herein.

The subject methods can be used on cells in vitro (e.g., in a cell-freesystem), in culture, e.g. in vitro or ex vivo. For example, IL-13receptor-expressing cells can be cultured in vitro in culture medium andthe contacting step can be effected by adding an IL-13 binding agent tothe culture medium. Alternatively, the method can be performed on cellspresent in a subject, e.g., as part of an in vivo (e.g., therapeutic orprophylactic) protocol. For example, the IL-13 binding agent can bedelivered locally or systemically.

The method can include contacting IL-13 with the IL-13 receptor complex,or subunit thereof, under conditions that allow an interaction betweenIL-13 and the IL-13 receptor complex, or subunit thereof, to occur tothereby form an IL-13/IL-13 receptor mixture. Generally, the IL-13binding agent is provided in an effective amount, e.g., so thatcontacting the IL-13/IL-13 receptor mixture modulates, e.g., interfereswith (e.g., inhibits, blocks or otherwise reduces) the interactionbetween IL-13 and the receptor protein or at least one function ofIL-13, e.g., IL-13 mediated signaling.

In another aspect, this application provides a method for detecting thepresence of IL-13 in a sample in vitro (e.g., a biological sample, suchas serum, plasma, tissue, biopsy). The subject method can be used todiagnose a disorder, e.g., an immune cell-associated disorder. Themethod includes: (i) contacting the sample or a control sample with anIL-13 binding agent, e.g., an anti-IL-13 antibody molecule, e.g., asdescribed herein; and (ii) detecting formation of a complex between theIL-13 binding agent and the sample or the control sample, wherein astatistically significant change in the formation of the complex in thesample relative to the control sample is indicative of the presence ofthe IL-13 in the sample.

In yet another aspect, this application provides a method for detectingthe presence of IL-13 in vivo (e.g., in vivo imaging in a subject). Thesubject method can be used to diagnose a disorder, e.g., anIL-13-associated disorder. The method includes: (i) administering anIL-13 binding agent, e.g., an anti-IL-13 antibody molecule, e.g., asdescribed herein, to a subject or a control subject under conditionsthat allow binding of the binding agent to IL-13; and (ii) detectingformation of a complex between the binding agent and IL-13, wherein astatistically significant change in the formation of the complex in thesubject relative to the control subject is indicative of the presence ofIL-13.

For example, the antibody molecule is directly or indirectly labeledwith a detectable substance to facilitate detection of the bound orunbound antibody. Suitable detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescent materialsand radioactive materials.

Methods for delivering or targeting an agent, e.g., a therapeutic or acytotoxic agent, to an IL-13-expressing cell in vivo are also disclosed.

In one aspect, the invention features a polypeptide that comprises thesequence, or a functional fragment thereof:

(SEQ ID NO: 14) SPVPPSTALKELIEELVNITQNQKAPLCNGSMVWSINLTAGVYCAALESLINVSGCSAIEKTQRMLNGFCPHKVSAGQFSSLRVRDTKIEVAQFVK DLLVHLKKLFREGQFN

The polypeptide can further include:

MALLLTMVIALTCLGGFASP, (SEQ ID NO: 127)e.g., as an N-terminal signal sequence. For example, the polypeptide isan IL-13 protein from cynomolgus monkey (herein, “NHP-IL-13”). The,NHP-IL-13 can be a mature IL-13 protein or an unprocessed full lengthIL-13 protein. Peptides of the above sequence, e.g., peptides thatdiffer from corresponding peptides in human IL-13, can be used, e.g., asan immunogen or target compound.

Also featured are related polypeptides that differ from human IL-13 atone or more of the boldfaced positions above but are identical to humanIL-13 at the non-boldface positions above. For example, one or more ofthe boldfaced positions can be an alanine, or a conservativesubstitution of the corresponding residue in the cynomolgus sequence(above) or the corresponding residue in the human sequence. Theinvention also features peptides, e.g., of at least 5 or 6 amino acidsfrom the above sequence. The peptides can be included in a heterologousprotein (e.g., a protein other than an IL-13), a chimeric protein (e.g.,a human IL-13) or can be in an isolated peptide, e.g., one that does notinclude other sequences. The peptides can also be fused or conjugated toother compounds, e.g., a carrier. In one embodiment, the peptideincludes at least one amino acid residue that differs from human IL-13.Exemplary peptides are described below.

Also featured are nucleic acids encoding the cynomolgus IL-13 sequenceand variants thereof. The polypeptide can be used to provide an IL-13binding agent that binds the cynomolgus monkey IL-13, and, optionally,also an IL-13 protein from another species, e.g., a human IL-13.

In one aspect, the invention features a method of providing a targetbinding molecule that specifically binds to a human target protein. Forexample, the target binding molecule is an antibody molecule. The methodincludes: providing a target protein that comprises at least a portionof a non-human protein, the portion being homologous to (at least 70,75, 80, 85, 87, 90, 92, 94, 95, 96, 97, or 98% identical to) acorresponding portion of a human target protein, but differing by atleast one amino acid (e.g., at least one, two, three, four, five, six,seven, eight, or nine amino acids); obtaining a binding agent thatspecifically binds to the antigen; and evaluating if the binding agentspecifically binds to the human target protein or evaluating efficacy ofthe binding agent in modulating activity of the human target protein.The method can further include administering the binding agent (e.g., anantibody molecule) or a derivative (e.g., a humanized antibody molecule)to a human subject. In one embodiment, the human target protein is acytokine, e.g., an interleukin, e.g., IL-13 or IL-4. The non-humanprotein can be from a non-human primate, e.g., a rhesus monkey, acynomolgus monkey, or a pigtail macaque.

In one embodiment, the step of obtaining comprises using a proteinexpression library, e.g., a phage or ribosome display library. Forexample, the library displays antibody molecules such as Fab's orscFv's. In one embodiment, the step of obtaining comprises immunizing ananimal using the antigen as an immunogen. For example, the animal can bea rodent, e.g., a mouse or rat. The animal can be a transgenic animalthat has at least one human immunoglobulin gene.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

DEFINITIONS

The term “IL-13 binding agent,” as used herein, refers to any compound,such as a protein (e.g., a multi-chain polypeptide, a polypeptide) or apeptide, that includes an interface that binds to an IL-13 protein,e.g., a mammalian IL-13, particularly a human or non-human primateIL-13. The binding agent generally binds with a Kd of less than 5×10⁻⁷M. An exemplary IL-13 binding agent is a protein that includes anantigen binding site, e.g., an antibody molecule.

As used herein, the term “antibody molecule” refers to a proteincomprising at least one immunoglobulin variable domain sequence. Theterm antibody molecule includes, for example, full-length, matureantibodies and antigen-binding fragments of an antibody. For example, anantibody molecule can include a heavy (H) chain variable domain sequence(abbreviated herein as VH), and a light (L) chain variable domainsequence (abbreviated herein as VL). In another example, an antibodymolecule includes two heavy (H) chain variable domain sequences and twolight (L) chain variable domain sequence, thereby forming two antigenbinding sites. Examples of antigen-binding fragments include: (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment, which consists of a VH domain; (vi) a camelid orcamelized variable domain; and (vii) a single chain Fv (scFv).

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (CDR),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the framework region and CDRs has beenprecisely defined by a number of methods (see, Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and theAbM definition used by Oxford Molecular's AbM antibody modellingsoftware. See, generally, e.g., Protein Sequence and Structure Analysisof Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.:Duebel, S, and Kontermann, R., Springer-Verlag, Heidelberg). Generally,unless specifically indicated, the following definitions are used: AbMdefinition of CDR1 of the heavy chain variable domain and Kabatdefinitions for the other CDRs. In addition, embodiments of theinvention described with respect to Kabat or AbM CDRs may also beimplemented using Chothia hypervariable loops. Each VH and VL typicallyincludes three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence which can form the structure of an immunoglobulinvariable domain. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may or may not include one, two, or more N- orC-terminal amino acids, or may include other alterations that arecompatible with formation of the protein structure.

The term “antigen-binding site” refers to the part of an IL-13 bindingagent that comprises determinants that form an interface that binds tothe IL-13, e.g., a mammalian IL-13, e.g., human or non-human primateIL-13, or an epitope thereof. With respect to proteins (or proteinmimetics), the antigen-binding site typically includes one or more loops(of at least four amino acids or amino acid mimics) that form aninterface that binds to IL-13. Typically, the antigen-binding site of anantibody molecule includes at least one or two CDRs, or more typicallyat least three, four, five or six CDRs.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope. Amonoclonal antibody can be made by hybridoma technology or by methodsthat do not use hybridoma technology (e.g., recombinant methods).

An “effectively human” protein is a protein that does not evoke aneutralizing antibody response, e.g., the human anti-murine antibody(HAMA) response. HAMA can be problematic in a number of circumstances,e.g., if the antibody molecule is administered repeatedly, e.g., intreatment of a chronic or recurrent disease condition. A HAMA responsecan make repeated antibody administration potentially ineffectivebecause of an increased antibody clearance from the serum (see, e.g.,Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)) and alsobecause of potential allergic reactions (see, e.g., LoBuglio et al.,Hybridoma, 5:5117-5123 (1986)).

The term “isolated” refers to a molecule that is substantially free ofits natural environment. For instance, an isolated protein issubstantially free of cellular material or other proteins from the cellor tissue source from which it is derived. The term refers topreparations where the isolated protein is sufficiently pure to beadministered as a therapeutic composition, or at least 70% to 80% (w/w)pure, more preferably, at least 80%-90% (w/w) pure, even morepreferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%,98%, 99%, or 100% (w/w) pure. A “separated” compound refers to acompound that is removed from at least 90% of at least one component ofa sample from which the compound was obtained. Any compound describedherein can be provided as an isolated or separated compound.

An “epitope” refers to the site on a target compound that is bound by abinding agent, e.g., an antibody molecule. An epitope can be a linear orconformational epitope, or a combination thereof. In the case where thetarget compound is a protein, for example, an epitope may refer to theamino acids that are bound by the binding agent. Overlapping epitopesinclude at least one common amino acid residue.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueousand nonaqueous methods are described in that reference and either can beused. Specific hybridization conditions referred to herein are asfollows: 1) low stringency hybridization conditions in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions); 2) mediumstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringencyhybridization conditions in 6×SSC at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very highstringency hybridization conditions are 0.5 M sodium phosphate, 7% SDSat 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.Very high stringency conditions (4) are the preferred conditions and theones that are used unless otherwise specified.

An “IL-13 associated disorder” is one in which IL-13 contributes to apathology or symptom of the disorder. Accordingly, an IL-13 bindingagent, e.g., an IL-13 binding agent that is an antagonist of one or moreIL-13 associated activities, can be used to treat or prevent thedisorder.

The term “IL-13” includes the full length unprocessed form of thecytokines known in the art as IL-13 (irrespective of species origin, andincluding mammalian, e.g., human and non-human primate IL-13) as well asmature, processed forms thereof, as well as any fragment (of at least 5amino acids) or variant of such cytokines. Positions within the IL-13sequence can be designated in accordance to the numbering for the fulllength, unprocessed human IL-13 sequence. For an exemplary full-lengthmonkey IL-13, see SEQ ID NO:24; for mature, processed monkey IL-13, seeSEQ ID NO:14; for full-length human IL-13, see SEQ ID NO:178, and formature, processed human IL-13, see SEQ ID NO:124. An exemplary sequenceis recited as follows:

(SEQ ID NO: 178) MALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN

For example, position 130 is a site of a common polymorphism.

Exemplary sequences of IL-13 receptor proteins (e.g., IL-13Rα1 andIL-13Rα2) are described, e.g., in Donaldson et al. (1998) J Immunol.161:2317-24; U.S. Pat. No. 6,214,559; U.S. Pat. No. 6,248,714; and U.S.Pat. No. 6,268,480.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an alignment of full-length human and cynomolgus monkeyIL-13, SEQ ID NO:178 and SEQ ID NO:24, respectively.

FIG. 1B is a list of exemplary peptides from cynomolgus monkey IL-13,(SEQ ID NOs:179-188, respectively).

FIG. 2 is a graph depicting the neutralization of NHP IL-13 activity byvarious IL-13 binding agents, as measured by percentage of CD23⁺monocytes (y-axis). Concentration of MJ2-7 (Δ), C65 (

), and sIL-13RI2-Fc () are indicated on the x-axis.

FIG. 3 is a graph depicting the neutralization of NHP IL-13 activity byMJ2-7 (murine; ) or humanized MJ2-7 v2.11 (∘). NHP IL-13 activity wasmeasured by phosphorylation of STAT6 (y-axis) as a function of antibodyconcentration (x-axis).

FIG. 4 is a graph depicting the neutralization of NHP IL-13 activity byMJ2-7 v2.11 (∘) or sIL-13RI2-Fc (

) NHP IL-13 activity was measured by phosphorylation of STAT6 (y-axis)as a function of antagonist concentration (x-axis).

FIG. 5 is a graph depicting the neutralization of NHP IL-13 activity byMJ2-7 (Δ), C65 (

), or sIL-13RI2-Fc (). NHP IL-13 activity was measured byphosphorylation of STAT6 (y-axis) as a function of antagonistconcentration (x-axis).

FIG. 6A is a graph depicting induction of tenascin production (y-axis)by native human IL-13 (x-axis).

FIG. 6B is a graph depicting the neutralization of NHP IL-13 activity byMJ2-7, as measured by inhibition of induction of tenascin production(y-axis) as a function of antibody concentration (x-axis).

FIG. 7 is a graph depicting binding of MJ2-7 or control antibodies toNHP-IL-13 bound to sIL-13RI2-Fc coupled to a SPR chip.

FIG. 8 is a graph depicting binding of varying concentrations (0.09-600nM) of NHP IL-13 to captured hMJ2-7 V2-11 antibody.

FIG. 9 is a graph depicting the neutralization of NHP IL-13 activity bymouse MJ2-7 () or humanized Version 1 (∘), Version 2 (

), or Version 3 (A) antibodies. NHP IL-13 activity was measured byphosphorylation of STAT6 (y-axis) as a function of antibodyconcentration (x-axis).

FIG. 10 is a graph depicting the neutralization of NHP IL-13 activity byantibodies including mouse MJ2-7 VH and VL (O), mouse VH and humanizedVersion 2 VL (Δ), or Version 2 VH and VL (

), NHP IL-13 activity was measured by phosphorylation of STAT6 (y-axis)as a function of antibody concentration (x-axis).

FIGS. 11A and 11B are graphs depicting inhibition of binding of IL-13 toimmobilized IL-13 receptor by MJ2-7 antibody, as measured by ELISA.Binding is depicted as absorbance at 450 nm (y-axis). Concentration ofMJ2-7 antibody is depicted on the x-axis. FIG. 11A depicts binding toIL-13RI1. FIG. 11B depicts binding to IL-13RI2.

FIG. 12 is an alignment of DPK18 germline amino acid sequence (SEQ IDNO:126) and humanized MJ2-7 Version 3 VL (SEQ ID NO:190).

FIG. 13A is an amino acid sequence (SEQ ID NO:124) of mature, processedhuman IL-13.

FIG. 13B is an amino acid sequence (SEQ ID NO:125) of human IL-13Rα1.

DETAILED DESCRIPTION

Binding agents (e.g., anti-IL13 antibody molecules) that bindspecifically to IL-13 and modulate the ability of IL-13 to interact withIL-13 receptors and signaling mediators are disclosed. The agents can beused to modulate (e.g., inhibit) one or more IL-13-associatedactivities. IL-13 binding agents, e.g., as described herein, can be usedto modulate one or more IL-13-associated activities, e.g., in vivo,e.g., to treat or prevent IL-13-mediated disorders (e.g., asthma, airwayinflammation, atopic disorders, allergic responses, eosinophilia,fibrosis, and IL-13 associated cancers).

Anti-IL-13 Antibody Molecules

Numerous methods are available for obtaining antibody molecules. Oneexemplary method includes screening protein expression libraries, e.g.,phage or ribosome display libraries. Phage display is described, forexample, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO93/01288; WO 92/01047; WO 92/09690; and WO 90/02809. In addition to theuse of display libraries, other methods can be used to obtain ananti-IL-13 antibody molecule. For example, an IL-13 protein or a peptidethereof can be used as an antigen in a non-human animal, e.g., a rodent,e.g., a mouse, hamster, or rat.

In one embodiment, the non-human animal includes at least a part of ahuman immunoglobulin gene. For example, it is possible to engineer mousestrains deficient in mouse antibody production with large fragments ofthe human Ig loci. Using the hybridoma technology, antigen-specificmonoclonal antibodies derived from the genes with the desiredspecificity may be produced and selected. See, e.g., XENOMOUSE™, Greenet al. (1994) Nature Genetics 7:13-21, US 2003-0070185, WO 96/34096,published Oct. 31, 1996, and PCT Application No. PCT/US96/05928, filedApr. 29, 1996.

In another embodiment, a monoclonal antibody is obtained from thenon-human animal, and then modified, e.g., humanized or deimmunized.Winter describes an exemplary CDR-grafting method that may be used toprepare the humanized antibodies described herein (U.S. Pat. No.5,225,539). All of the CDRs of a particular human antibody may bereplaced with at least a portion of a non-human CDR, or only some of theCDRs may be replaced with non-human CDRs. It is only necessary toreplace the number of CDRs required for binding of the humanizedantibody to a predetermined antigen.

Humanized antibodies can be generated by replacing sequences of the Fvvariable domain that are not directly involved in antigen binding withequivalent sequences from human Fv variable domains. Exemplary methodsfor generating humanized antibody molecules are provided by Morrison(1985) Science 229:1202-1207; by Oi et al. (1986) BioTechniques 4:214;and by U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; U.S. Pat. No. 5,859,205; and U.S. Pat. No. 6,407,213. Thosemethods include isolating, manipulating, and expressing the nucleic acidsequences that encode all or part of immunoglobulin Fv variable domainsfrom at least one of a heavy or light chain. Such nucleic acids may beobtained from a hybridoma producing an antibody against a predeterminedtarget, as described above, as well as from other sources. Therecombinant DNA encoding the humanized antibody molecule can then becloned into an appropriate expression vector.

An IL-13-binding antibody molecule may also be modified by specificdeletion of human T cell epitopes or “deimmunization” by the methodsdisclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy and lightchain variable domains of an antibody can be analyzed for peptides thatbind to MHC Class II; these peptides represent potential T-cell epitopes(as defined in WO 98/52976 and WO 00/34317). For detection of potentialT-cell epitopes, a computer modeling approach termed “peptide threading”can be applied, and in addition a database of human MHC class II bindingpeptides can be searched for motifs present in the V_(H) and V_(L)sequences, as described in WO 98/52976 and WO 00/34317. These motifsbind to any of the 18 major MHC class II DR allotypes, and thusconstitute potential T cell epitopes. Potential T-cell epitopes detectedcan be eliminated by substituting small numbers of amino acid residuesin the variable domains, or preferably, by single amino acidsubstitutions. Typically, conservative substitutions are made. Often,but not exclusively, an amino acid common to a position in humangermline antibody sequences may be used. Human germline sequences, e.g.,are disclosed in Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798;Cook, G. P. et al. (1995) Immunol. Today Vol. 16 (5): 237-242; Chothia,D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995)EMBO J. 14:4628-4638. The V BASE directory provides a comprehensivedirectory of human immunoglobulin variable region sequences (compiled byTomlinson, I. A. et al. MRC Centre for Protein Engineering, Cambridge,UK). These sequences can be used as a source of human sequence, e.g.,for framework regions and CDRs. Consensus human framework regions canalso be used, e.g., as described in U.S. Pat. No. 6,300,064.

Additionally, chimeric, humanized, and single-chain antibody molecules(e.g., proteins that include both human and nonhuman portions), may beproduced using standard recombinant DNA techniques. Humanized antibodiesmay also be produced, for example, using transgenic mice that expresshuman heavy and light chain genes, but are incapable of expressing theendogenous mouse immunoglobulin heavy and light chain genes.

Additionally, the antibody molecules described herein also include thosethat bind to IL-13, interfere with the formation of a functional IL-13signaling complex, and have mutations in the constant regions of theheavy chain. It is sometimes desirable to mutate and inactivate certainfragments of the constant region. For example, mutations in the heavyconstant region can be made to produce antibodies with reduced bindingto the Fc receptor (FcR) and/or complement; such mutations are wellknown in the art. An example of such a mutation to the amino sequence ofthe constant region of the heavy chain of IgG is provided in SEQ IDNO:128. Certain active fragments of the CL and CH subunits (e.g., CH1)are covalently link to each other. A further aspect provides a methodfor obtaining an antigen-binding site that is specific for a surface ofIL-13 that participates in forming a functional IL-13 signaling complex.

Exemplary antibody molecules can include sequences of VL chains as setforth in SEQ ID NOs:30-46, and/or of VH chains as set forth in and SEQID NOs:50-115, but also can include variants of these sequences thatretain IL-13 binding ability. Such variants may be derived from theprovided sequences using techniques well known in the art. Amino acidsubstitutions, deletions, or additions, can be made in either the FRs orin the CDRs. Whereas changes in the framework regions are usuallydesigned to improve stability and reduce immunogenicity of the antibodymolecule, changes in the CDRs are usually designed to increase affinityof the antibody molecule for its target. Such affinity-increasingchanges are typically determined empirically by altering the CDR regionand testing the antibody molecule. Such alterations can be madeaccording to the methods described in Antibody Engineering, 2nd. ed.(1995), ed. Borrebaeck, Oxford University Press.

An exemplary method for obtaining a heavy chain variable domain sequencethat is a variant of a heavy chain variable domain sequence describedherein, includes adding, deleting, substituting, or inserting one ormore amino acids in a heavy chain variable domain sequence describedherein, optionally combining the heavy chain variable domain sequencewith one or more light chain variable domain sequences, and testing aprotein that includes the modified heavy chain variable domain sequencefor specific binding to IL-13, and (preferably) testing the ability ofsuch antigen-binding domain to modulate one or more IL-13-associatedactivities. An analogous method may be employed using one or moresequence variants of a light chain variable domain sequence describedherein.

Variants of antibody molecules can be prepared by creating librarieswith one or more varied CDRs and screening the libraries to find membersthat bind to IL-13, e.g., with improved affinity. For example, Marks etal. (Bio/Technology (1992) 10:779-83) describe methods of producingrepertoires of antibody variable domains in which consensus primersdirected at or adjacent to the 5′ end of the variable domain area areused in conjunction with consensus primers to the third framework regionof human VH genes to provide a repertoire of VH variable domains lackinga CDR3. The repertoire may be combined with a CDR3 of a particularantibody. Further, the CDR3-derived sequences may be shuffled withrepertoires of VH or VL domains lacking a CDR3, and the shuffledcomplete VH or VL domains combined with a cognate VL or VH domain toprovide specific antigen-binding fragments. The repertoire may then bedisplayed in a suitable host system such as the phage display system ofWO 92/01047, so that suitable antigen-binding fragments can be selected.Analogous shuffling or combinatorial techniques are also disclosed byStemmer (Nature (1994) 370:389-91). A further alternative is to generatealtered VH or VL regions using random mutagenesis of one or moreselected VH and/or VL genes to generate mutations within the entirevariable domain. See, e.g., Gram et al. Proc. Nat. Acad. Sci. USA (1992)89:3576-80.

Another method that may be used is to direct mutagenesis to CDR regionsof VH or VL genes. Such techniques are disclosed by, e.g., Barbas et al.(Proc. Nat. Acad. Sci. USA (1994) 91:3809-13) and Schier et al. (J. Mol.Biol. (1996) 263:551-67). Similarly, one or more, or all three CDRs maybe grafted into a repertoire of VH or VL domains, or even some otherscaffold (such as a fibronectin domain). The resulting protein isevaluated for ability to bind to IL-13.

In one embodiment, a binding agent that binds to a target is modified,e.g., by mutagenesis, to provide a pool of modified binding agents. Themodified binding agents are then evaluated to identify one or morealtered binding agents which have altered functional properties (e.g.,improved binding, improved stability, lengthened stability in vivo). Inone implementation, display library technology is used to select orscreen the pool of modified binding agents. Higher affinity bindingagents are then identified from the second library, e.g., by usinghigher stringency or more competitive binding and washing conditions.Other screening techniques can also be used.

In some embodiments, the mutagenesis is targeted to regions known orlikely to be at the binding interface. If, for example, the identifiedbinding agents are antibody molecules, then mutagenesis can be directedto the CDR regions of the heavy or light chains as described herein.Further, mutagenesis can be directed to framework regions near oradjacent to the CDRs, e.g., framework regions, particular within 10, 5,or 3 amino acids of a CDR junction. In the case of antibodies,mutagenesis can also be limited to one or a few of the CDRs, e.g., tomake step-wise improvements.

In one embodiment, mutagenesis is used to make an antibody more similarto one or more germline sequences. One exemplary germlining method caninclude: identifying one or more germline sequences that are similar(e.g., most similar in a particular database) to the sequence of theisolated antibody. Then mutations (at the amino acid level) can be madein the isolated antibody, either incrementally, in combination, or both.For example, a nucleic acid library that includes sequences encodingsome or all possible germline mutations is made. The mutated antibodiesare then evaluated, e.g., to identify an antibody that has one or moreadditional germline residues relative to the isolated antibody and thatis still useful (e.g., has a functional activity). In one embodiment, asmany germline residues are introduced into an isolated antibody aspossible.

In one embodiment, mutagenesis is used to substitute or insert one ormore germline residues into a CDR region. For example, the germline CDRresidue can be from a germline sequence that is similar (e.g., mostsimilar) to the variable domain being modified. After mutagenesis,activity (e.g., binding or other functional activity) of the antibodycan be evaluated to determine if the germline residue or residues aretolerated. Similar mutagenesis can be performed in the frameworkregions.

Selecting a germline sequence can be performed in different ways. Forexample, a germline sequence can be selected if it meets a predeterminedcriteria for selectivity or similarity, e.g., at least a certainpercentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 99.5% identity. The selection can be performed usingat least 2, 3, 5, or 10 germline sequences. In the case of CDR1 andCDR2, identifying a similar germline sequence can include selecting onesuch sequence. In the case of CDR3, identifying a similar germlinesequence can include selecting one such sequence, but may includingusing two germline sequences that separately contribute to theamino-terminal portion and the carboxy-terminal portion. In otherimplementations more than one or two germline sequences are used, e.g.,to form a consensus sequence.

In other embodiments, the antibody may be modified to have an alteredglycosylation pattern (i.e., altered from the original or nativeglycosylation pattern). As used in this context, “altered” means havingone or more carbohydrate moieties deleted, and/or having one or moreglycosylation sites added to the original antibody. Addition ofglycosylation sites to the presently disclosed antibodies may beaccomplished by altering the amino acid sequence to containglycosylation site consensus sequences; such techniques are well knownin the art. Another means of increasing the number of carbohydratemoieties on the antibodies is by chemical or enzymatic coupling ofglycosides to the amino acid residues of the antibody. These methods aredescribed in, e.g., WO 87/05330, and Aplin and Wriston (1981) CRC Crit.Rev. Biochem. 22:259-306. Removal of any carbohydrate moieties presenton the antibodies may be accomplished chemically or enzymatically asdescribed in the art (Hakimuddin et al. (1987) Arch. Biochem. Biophys.259:52; Edge et al. (1981) Anal. Biochem. 118:131; and Thotakura et al.(1987) Meth. Enzymol. 138:350). See, e.g., U.S. Pat. No. 5,869,046 for amodification that increases in vivo half life by providing a salvagereceptor binding epitope.

In one embodiment, an antibody molecule has CDR sequences that differonly insubstantially from those of MJ 2-7 or C65. Insubstantialdifferences include minor amino acid changes, such as substitutions of 1or 2 out of any of typically 5-7 amino acids in the sequence of a CDR,e.g., a Chothia or Kabat CDR. Typically, an amino acid is substituted bya related amino acid having similar charge, hydrophobic, orstereochemical characteristics. Such substitutions are within theordinary skills of an artisan. Unlike in CDRs, more substantial changesin structure framework regions (FRs) can be made without adverselyaffecting the binding properties of an antibody. Changes to FRs include,but are not limited to, humanizing a nonhuman-derived framework orengineering certain framework residues that are important for antigencontact or for stabilizing the binding site, e.g., changing the class orsubclass of the constant region, changing specific amino acid residueswhich might alter an effector function such as Fc receptor binding (Lundet al. (1991) J. Immunol. 147:2657-62; Morgan et al. (1995) Immunology86:319-24), or changing the species from which the constant region isderived. Antibodies may have mutations in the CH2 region of the heavychain that reduce or alter effector function, e.g., Fc receptor bindingand complement activation. For example, antibodies may have mutationssuch as those described in U.S. Pat. Nos. 5,624,821 and 5,648,260. Inthe IgG1 or IgG2 heavy chain, for example, such mutations may be made toresemble the amino acid sequence set forth in SEQ ID NO:17. Antibodiesmay also have mutations that stabilize the disulfide bond between thetwo heavy chains of an immunoglobulin, such as mutations in the hingeregion of IgG4, as disclosed in the art (e.g., Angal et al. (1993) Mol.Immunol. 30:105-08).

The IL-13 binding agents can be in the form of intact antibodies,antigen-binding fragments of antibodies, e.g., Fab, F(ab′)₂, Fd, dAb,and scFv fragments, and intact antibodies and fragments that have beenmutated either in their constant and/or variable domain (e.g., mutationsto produce chimeric, partially humanized, or fully humanized antibodies,as well as to produce antibodies with a desired trait, e.g., enhancedIL-13 binding and/or reduced FcR binding).

Antibody Production. Some antibody molecules, e.g., Fabs, can beproduced in bacterial cells, e.g., E. coli cells. For example, if theFab is encoded by sequences in a phage display vector that includes asuppressible stop codon between the display entity and a bacteriophageprotein (or fragment thereof), the vector nucleic acid can betransferred into a bacterial cell that cannot suppress a stop codon. Inthis case, the Fab is not fused to the gene III protein and is secretedinto the periplasm and/or media.

Antibody molecules can also be produced in eukaryotic cells. In oneembodiment, the antibodies (e.g., scFv's) are expressed in a yeast cellsuch as Pichia (see, e.g., Powers et al. (2001) J Immunol Methods.251:123-35), Hanseula, or Saccharomyces.

In one preferred embodiment, antibody molecules are produced inmammalian cells. Preferred mammalian host cells for expressing the cloneantibodies or antigen-binding fragments thereof include Chinese HamsterOvary (CHO cells) (including dhfr⁻ CHO cells, described in Urlaub andChasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFRselectable marker, e.g., as described in Kaufman and Sharp (1982) Mol.Biol. 159:601-621), lymphocytic cell lines, e.g., NS0 myeloma cells andSP2 cells, COS cells, and a cell from a transgenic animal, e.g., atransgenic mammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequences encoding the antibodymolecule, the recombinant expression vectors may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin, or methotrexate, on a host cell into which the vector hasbeen introduced.

In an exemplary system for recombinant expression of an antibodymolecule, a recombinant expression vector encoding both the antibodyheavy chain and the antibody light chain is introduced into dhfr⁻ CHOcells by calcium phosphate-mediated transfection. Within the recombinantexpression vector, the antibody heavy and light chain genes are eachoperatively linked to enhancer/promoter regulatory elements (e.g.,derived from SV40, CMV, adenovirus and the like, such as a CMVenhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLPpromoter regulatory element) to drive high levels of transcription ofthe genes. The recombinant expression vector also carries a DHFR gene,which allows for selection of CHO cells that have been transfected withthe vector using methotrexate selection/amplification. The selectedtransformant host cells can be cultured to allow for expression of theantibody heavy and light chains and intact antibody is recovered fromthe culture medium. Standard molecular biology techniques can be used toprepare the recombinant expression vector, transfect the host cells,select for transformants, culture the host cells and recover theantibody molecule from the culture medium. For example, some antibodymolecules can be isolated by affinity chromatography with a Protein A orProtein G coupled matrix.

For antibody molecules that include an Fc domain, the antibodyproduction system preferably synthesizes antibodies in which the Fcregion is glycosylated. For example, the Fc domain of IgG molecules isglycosylated at asparagine 297 in the CH2 domain. This asparagine is thesite for modification with biantennary-type oligosaccharides. It hasbeen demonstrated that this glycosylation is required for effectorfunctions mediated by Fcγ receptors and complement Clq (Burton and Woof(1992) Adv. Immunol. 51:1-84; Jefferis et al. (1998) Immunol. Rev.163:59-76). In one embodiment, the Fc domain is produced in a mammalianexpression system that appropriately glycosylates the residuecorresponding to asparagine 297. The Fc domain can also include othereukaryotic post-translational modifications.

Antibody molecules can also be produced by a transgenic animal. Forexample, U.S. Pat. No. 5,849,992 describes a method of expressing anantibody in the mammary gland of a transgenic mammal. A transgene isconstructed that includes a milk-specific promoter and nucleic acidsencoding the antibody molecule and a signal sequence for secretion. Themilk produced by females of such transgenic mammals includes,secreted-therein, the antibody of interest. The antibody molecule can bepurified from the milk, or for some applications, used directly.

Characterization

The binding properties of a binding agent may be measured by any method,e.g., one of the following methods: BIACORE™ analysis, Enzyme LinkedImmunosorbent Assay (ELISA), x-ray crystallography, sequence analysisand scanning mutagenesis. The ability of a protein to neutralize and/orinhibit one or more IL-13-associated activities may be measured by thefollowing methods: assays for measuring the proliferation of an IL-13dependent cell line, e.g. TFI; assays for measuring the expression ofIL-13-mediated polypeptides, e.g., flow cytometric analysis of theexpression of CD23; assays evaluating the activity of downstreamsignaling molecules, e.g., STATE; assays evaluating production oftenascin; assays testing the efficiency of an antibody described hereinto prevent asthma in a relevant animal model, e.g., the cynomolgusmonkey, and other assays. An IL-13 binding agent, particularly an IL-13antibody molecule, can have a statistically significant effect in one ormore of these assays. Exemplary assays for binding properties includethe following.

The binding interaction of a IL-13 binding agent and a target (e.g.,IL-13) can be analyzed using surface plasmon resonance (SPR). SPR orBiomolecular Interaction Analysis (BIA) detects biospecific interactionsin real time, without labeling any of the interactants. Changes in themass at the binding surface (indicative of a binding event) of the BIAchip result in alterations of the refractive index of light near thesurface. The changes in the refractivity generate a detectable signal,which are measured as an indication of real-time reactions betweenbiological molecules. Methods for using SPR are described, for example,in U.S. Pat. No. 5,641,640; Raether (1988) Surface Plasmons SpringerVerlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345; Szaboet al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line resourcesprovide by BIAcore International AB (Uppsala, Sweden).

Information from SPR can be used to provide an accurate and quantitativemeasure of the equilibrium dissociation constant (K_(d)), and kineticparameters, including K_(on) and K_(off), for the binding of a moleculeto a target. Such data can be used to compare different molecules.Information from SPR can also be used to develop structure-activityrelationships (SAR). For example, the kinetic and equilibrium bindingparameters of different antibody molecule can be evaluated. Variantamino acids at given positions can be identified that correlate withparticular binding parameters, e.g., high affinity and slow K_(off).This information can be combined with structural modeling (e.g., usinghomology modeling, energy minimization, or structure determination byx-ray crystallography or NMR). As a result, an understanding of thephysical interaction between the protein and its target can beformulated and used to guide other design processes.

Respiratory Disorders

IL-13 binding agents, e.g., anti-IL-13 antibody molecules, can be usedto treat or prevent respiratory disorders including, but are not limitedto asthma (e.g., allergic and nonallergic asthma (e.g., due toinfection, e.g., with respiratory syncytial virus (RSV), e.g., inyounger children)); bronchitis (e.g., chronic bronchitis); chronicobstructive pulmonary disease (COPD) (e.g., emphysema (e.g.,cigarette-induced emphysema); conditions involving airway inflammation,eosinophilia, fibrosis and excess mucus production, e.g., cysticfibrosis, pulmonary fibrosis, and allergic rhinitis. For example, anIL-13 binding agent (e.g., an anti-IL-13 antibody molecule) can beadministered in an amount effective to treat or prevent the disorder orto ameliorate at least one symptom of the disorder.

Asthma can be triggered by myriad conditions, e.g., inhalation of anallergen, presence of an upper-respiratory or ear infection, etc.(Opperwall (2003) Nurs. Clin. North Am. 38:697-711). Allergic asthma ischaracterized by airway hyperresponsiveness (AHR) to a variety ofspecific and nonspecific stimuli, elevated serum immunoglobulin E (IgE),excessive airway mucus production, edema, and bronchial epithelialinjury (Wills-Karp, supra). Allergic asthma begins when the allergenprovokes an immediate early airway response, which is frequentlyfollowed several hours later by a delayed late-phase airway response(LAR) (Henderson et al. (2000) J. Immunol. 164:1086-95). During LAR,there is an influx of eosinophils, lymphocytes, and macrophagesthroughout the airway wall and the bronchial fluid. (Henderson et al.,supra). Lung eosinophilia is a hallmark of allergic asthma and isresponsible for much of the damage to the respiratory epithelium (Li etal. (1999) J. Immunol. 162:2477-87).

CD4⁺ T helper (Th) cells are important for the chronic inflammationassociated with asthma (Henderson et al., supra). Several studies haveshown that commitment of CD4+ cells to type 2 T helper (Th2) cells andthe subsequent production of type 2 cytokines (e.g., IL-4, IL-5, IL-10,and IL-13) are important in the allergic inflammatory response leadingto AHR (Tomkinson et al. (2001) J. Immunol. 166:5792-5800, andreferences cited therein). First, CD4⁺ T cells have been shown to benecessary for allergy-induced asthma in murine models. Second, CD4⁺ Tcells producing type 2 cytokines undergo expansion not only in theseanimal models but also in patients with allergic asthma. Third, type 2cytokine levels are increased in the airway tissues of animal models andasthmatics. Fourth, Th2 cytokines have been implicated as playing acentral role in eosinophil recruitment in murine models of allergicasthma, and adoptively transferred Th2 cells have been correlated withincreased levels of eotaxin (a potent eosinophil chemoattractant) in thelung as well as lung eosinophilia (Wills-Karp et al., supra; Li et al.,supra).

The methods for treating or preventing asthma described herein includethose for extrinsic asthma (also known as allergic asthma or atopicasthma), intrinsic asthma (also known as non-allergic asthma ornon-atopic asthma) or combinations of both, which has been referred toas mixed asthma. Extrinsic or allergic asthma includes incidents causedby, or associated with, e.g., allergens, such as pollens, spores,grasses or weeds, pet danders, dust, mites, etc. As allergens and otherirritants present themselves at varying points over the year, thesetypes of incidents are also referred to as seasonal asthma. Alsoincluded in the group of extrinsic asthma is bronchial asthma andallergic bronchopulmonary aspergillosis.

Disorders that can be treated or alleviated by the agents describedherein include those respiratory disorders and asthma caused byinfectious agents, such as viruses (e.g., cold and flu viruses,respiratory syncytial virus (RSV), paramyxovirus, rhinovirus andinfluenza viruses. RSV, rhinovirus and influenza virus infections arecommon in children, and are one leading cause of respiratory tractillnesses in infants and young children. Children with viralbronchiolitis can develop chronic wheezing and asthma, which can betreated using the methods described herein. Also included are the asthmaconditions which may be brought about in some asthmatics by exerciseand/or cold air. The methods are useful for asthmas associated withsmoke exposure (e.g., cigarette-induced and industrial smoke), as wellas industrial and occupational exposures, such as smoke, ozone, noxiousgases, sulfur dioxide, nitrous oxide, fumes, including isocyanates, frompaint, plastics, polyurethanes, varnishes, etc., wood, plant or otherorganic dusts, etc. The methods are also useful for asthmatic incidentsassociated with food additives, preservatives or pharmacological agents.Also included are methods for treating, inhibiting or alleviating thetypes of asthma referred to as silent asthma or cough variant asthma.

The methods disclosed herein are also useful for treatment andalleviation of asthma associated with gastroesophageal reflux (GERD),which can stimulate bronchoconstriction. GERD, along with retainedbodily secretions, suppressed cough, and exposure to allergens andirritants in the bedroom can contribute to asthmatic conditions and havebeen collectively referred to as nighttime asthma or nocturnal asthma.In methods of treatment, inhibition or alleviation of asthma associatedwith GERD, a pharmaceutically effective amount of the IL-13 bindingagent can be used as described herein in combination with apharmaceutically effective amount of an agent for treating GERD. Theseagents include, but are not limited to, proton pump inhibiting agentslike PROTONIX® brand of delayed-release pantoprazole sodium tablets,PRILOSEC® brand omeprazole delayed release capsules, ACIPHEX® brandrebeprazole sodium delayed release tablets or PREVACID® brand delayedrelease lansoprazole capsules.

Atopic Disorders and Symptoms Thereof

It has been observed that cells from atopic patients have enhancedsensitivity to IL-13. Accordingly, an IL-13 binding agent (e.g., anIL-13 binding agent such as an antibody molecule described herein) canbe administered in an amount effective to treat or prevent an atopicdisorder. “Atopic” refers to a group of diseases in which there is oftenan inherited tendency to develop an allergic reaction.

Examples of atopic disorders include allergy, allergic rhinitis, atopicdermatitis, asthma and hay fever. Asthma is a phenotypicallyheterogeneous disorder associated with intermittent respiratory symptomssuch as, e.g., bronchial hyperresponsiveness and reversible airflowobstruction. Immunohistopathologic features of asthma include, e.g.,denudation of airway epithelium, collagen deposition beneath thebasement membrane; edema; mast cell activation; and inflammatory cellinfiltration (e.g., by neutrophils, eosinophils, and lymphocytes).Airway inflammation can further contribute to airwayhyperresponsiveness, airflow limitation, acute bronchoconstriction,mucus plug formation, airway wall remodeling, and other respiratorysymptoms. An IL-13 binding agent (e.g., an IL-13 binding agent such asan antibody molecule described herein) can be administered in an amounteffective to ameliorate one or more of these symptoms.

Symptoms of allergic rhinitis (hay fever) include itchy, runny,sneezing, or stuffy nose, and itchy eyes. An IL-13 binding agent can beadministered to ameliorate one or more of these symptoms. Atopicdermatitis is a chronic (long-lasting) disease that affects the skin.Information about atopic dermatitis is available, e.g., from NIHPublication No. 03-4272. In atopic dermatitis, the skin can becomeextremely itchy, leading to redness, swelling, cracking, weeping clearfluid, and finally, crusting and scaling. In many cases, there areperiods of time when the disease is worse (called exacerbations orflares) followed by periods when the skin improves or clears up entirely(called remissions). Atopic dermatitis is often referred to as “eczema,”which is a general term for the several types of inflammation of theskin. Atopic dermatitis is the most common of the many types of eczema.Examples of atopic dermatitis include: allergic contact eczema(dermatitis: a red, itchy, weepy reaction where the skin has come intocontact with a substance that the immune system recognizes as foreign,such as poison ivy or certain preservatives in creams and lotions);contact eczema (a localized reaction that includes redness, itching, andburning where the skin has come into contact with an allergen (anallergy-causing substance) or with an irritant such as an acid, acleaning agent, or other chemical); dyshidrotic eczema (irritation ofthe skin on the palms of hands and soles of the feet characterized byclear, deep blisters that itch and burn); neurodermatitis (scaly patchesof the skin on the head, lower legs, wrists, or forearms caused by alocalized itch (such as an insect bite) that become intensely irritatedwhen scratched); nummular eczema (coin-shaped patches of irritatedskin-most common on the arms, back, buttocks, and lower legs-that may becrusted, scaling, and extremely itchy); seborrheic eczema (yellowish,oily, scaly patches of skin on the scalp, face, and occasionally otherparts of the body). Additional particular symptoms include stasisdermatitis, atopic pleat (Dennie-Morgan fold), cheilitis, hyperlinearpalms, hyperpigmented eyelids (eyelids that have become darker in colorfrom inflammation or hay fever), ichthyosis, keratosis pilaris,lichenification, papules, and urticaria. An IL-13 binding agent can beadministered to ameliorate one or more of these symptoms.

An exemplary method for treating allergic rhinitis or other allergicdisorder can include initiating therapy with an IL-13 binding agentprior to exposure to an allergen, e.g., prior to seasonal exposure to anallergen, e.g., prior to allergen blooms. Such therapy can include oneor more doses, e.g., doses at regular intervals.

Cancer

IL-13 and its receptors may be involved in the development of at leastsome types of cancer, e.g., a cancer derived from hematopoietic cells ora cancer derived from brain or neuronal cells (e.g., a glioblastoma).For example, blockade of the IL-13 signaling pathway, e.g., via use of asoluble IL-13 receptor or a STATE −/− deficient mouse, leads to delayedtumor onset and/or growth of Hodgkins lymphoma cell lines or ametastatic mammary carcinoma, respectively (Trieu et al. (2004) CancerRes. 64: 3271-75; Ostrand-Rosenberg et al. (2000) J. Immunol. 165:6015-6019). Cancers that express IL-13R(2 (Husain and Puri (2003) J.Neurooncol. 65:37-48; Mintz et al. (2003) J. Neurooncol. 64:117-23) canbe specifically targeted by anti-IL-13 antibodies described herein.IL-13 binding agents, e.g., anti-IL-13 antibody molecules, can be usefulto inhibit cancer cell proliferation or other cancer cell activity. Acancer refers to one or more cells that has a loss of responsiveness tonormal growth controls, and typically proliferates with reducedregulation relative to a corresponding normal cell.

Examples of cancers against which IL-13 binding agents (e.g., an IL-13binding agent such as an antibody or antigen binding fragment describedherein) can be used for treatment include leukemias, e.g., B-cellchronic lymphocytic leukemia, acute myelogenous leukemia, and humanT-cell leukemia virus type 1 (HTLV-1) transformed T cells; lymphomas,e.g. T cell lymphoma, Hodgkin's lymphoma; glioblastomas; pancreaticcancers; renal cell carcinoma; ovarian carcinoma; and AIDS-Kaposi'ssarcoma. For example, an IL-13 binding agent (e.g., an anti-IL-13antibody molecule) can be administered in an amount effective to treator prevent the disorder, e.g., to reduce cell proliferation, or toameliorate at least one symptom of the disorder.

Fibrosis

IL-13 binding agents can also be useful in treating inflammation andfibrosis, e.g., fibrosis of the liver. IL-13 production has beencorrelated with the progression of liver inflammation (e.g., viralhepatitis) toward cirrhosis, and possibly, hepatocellular carcinoma (deLalla et al. (2004) J. Immunol. 173:1417-1425). Fibrosis occurs, e.g.,when normal tissue is replaced by scar tissue, often followinginflammation. Hepatitis B and hepatitis C viruses both cause a fibroticreaction in the liver, which can progress to cirrhosis. Cirrhosis, inturn, can evolve into severe complications such as liver failure orhepatocellular carcinoma. Blocking IL-13 activity using the IL-13binding agents, e.g., anti-IL-13 antibodies, described herein can reduceinflammation and fibrosis, e.g., the inflammation, fibrosis, andcirrhosis associated with liver diseases, especially hepatitis B and C.For example, an IL-13 binding agent (e.g., an anti-IL-13 antibodymolecule) can be administered in an amount effective to treat or preventthe disorder or to ameliorate at least one symptom of the inflammatoryand/or fibrotic disorder.

Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is the general name for diseases thatcause inflammation of the intestines. Two examples of inflammatory boweldisease are Crohn's disease and ulcerative colitis. IL-13/STATEsignaling has been found to be involved in inflammation-inducedhypercontractivity of mouse smooth muscle, a model of inflammatory boweldisease (Akiho et al. (2002) Am. J. Physiol. Gastrointest. LiverPhysiol. 282:G226-232). For example, an IL-13 binding agent (e.g., ananti-IL-13 antibody molecule) can be administered in an amount effectiveto treat or prevent the disorder or to ameliorate at least one symptomof the inflammatory bowel disorder.

Additional IL-13 Binding Agents

Also provided are binding agents, other than binding agents that areantibodies and fragments thereof, that bind to IL-13, particularlybinding agents that compete with MJ2-7 or C65 and other antibodiesdescribed herein for binding to IL-13. For example, the binding agentscan bind to the same epitope or an overlapping epitope as MJ2-7 or C65on IL-13. The binding agents preferably inhibit or neutralize IL-13activity. For example, the binding agents inhibit binding of IL-13 toIL13Rα1 and, e.g., does not prevent binding of IL-13 to IL-4Rα. Suchbinding agents can be used in the methods described herein, e.g., themethods of treating and preventing disorders. All embodiments describedherein can be adapted for use with IL-13 binding agents.

Binding agents can be identified by a number of means, includingmodifying a variable domain described herein or grafting one or moreCDRs of a variable domain described herein onto another scaffold domain.Binding agents can also be identified from diverse libraries, e.g., byscreening. One method for screening protein libraries uses phagedisplay. Particular regions of a protein are varied and proteins thatinteract with IL-13 are identified, e.g., by retention on a solidsupport or by other physical association. To identify particular bindingagents that bind to the same epitope or an overlapping epitope as MJ2-7or C65 on IL-13, binding agents can be eluted by adding MJ2-7 or C65 (orrelated antibody), or binding agents can be evaluated in competitionexperiments with MJ2-7 or C65 (or related antibody). It is also possibleto deplete the library of agents that bind to other epitopes bycontacting the library to a complex that contains IL-13 and MJ2-7 or C65(or related antibody). The depleted library can then be contacted toIL-13 to obtain a binding agent that binds to IL-13 but not to IL-13when it is bound by MJ 2-7 or C65. It is also possible to use peptidesfrom IL-13 that contain the MJ 2-7 or C65 epitope as a target.

Phage display is described, for example, in U.S. Pat. No. 5,223,409;Smith (1985) Science 228:1315-1317; WO 92/18619; WO 91/17271; WO92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO90/02809; WO 94/05781; Fuchs et al. (1991) Bio/Technology 9:1370-1372;Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989)Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734;Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991)Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al.(1991) Bio/Technology 9:1373-1377; Rebar et al. (1996) Methods Enzymol.267:129-49; and Barbas et al. (1991) PNAS 88:7978-7982. Yeast surfacedisplay is described, e.g., in Boder and Wittrup (1997) Nat. Biotechnol.15:553-557. Another form of display is ribosome display. See, e.g.,Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes etal. (2000) Nat. Biotechnol. 18:1287-92; Hanes et al. (2000) MethodsEnzymol. 328:404-30. and Schaffitzel et al. (1999) J Immunol Methods.231(1-2):119-35.

Binding agents that bind to IL-13 can have structural features of onescaffold proteins, e.g., a folded domain. An exemplary scaffold domain,based on an antibody, is a “minibody” scaffold has been designed bydeleting three beta strands from a heavy chain variable domain of amonoclonal antibody (Tramontano et al., 1994, J. Mol. Recognit. 7:9; andMartin et al., 1994, EMBO J. 13:5303-5309). This domain includes 61residues and can be used to present two hypervariable loops, e.g., oneor more hypervariable loops of a variable domain described herein or avariant described herein. In another approach, the binding agentincludes a scaffold domain that is a V-like domain (Coia et al. WO99/45110). V-like domains refer to a domain that has similar structuralfeatures to the variable heavy (VH) or variable light (VL) domains ofantibodies. Another scaffold domain is derived from tendamistatin, a 74residue, six-strand beta sheet sandwich held together by two disulfidebonds (McConnell and Hoess, 1995, J. Mol. Biol. 250:460). This parentprotein includes three loops. The loops can be modified (e.g., usingCDRs or hypervariable loops described herein) or varied, e.g., to selectdomains that bind to IL-13. WO 00/60070 describes a β-sandwich structurederived from the naturally occurring extracellular domain of CTLA-4 thatcan be used as a scaffold domain.

Still another scaffold domain for an IL-13 binding agent is a domainbased on the fibronectin type III domain or related fibronectin-likeproteins. The overall fold of the fibronectin type III (Fn3) domain isclosely related to that of the smallest functional antibody fragment,the variable domain of the antibody heavy chain. Fn3 is a β-sandwichsimilar to that of the antibody VH domain, except that Fn3 has sevenβ-strands instead of nine. There are three loops at the end of Fn3; thepositions of BC, DE and FG loops approximately correspond to those ofCDR1, 2 and 3 of the VH domain of an antibody. Fn3 is advantageousbecause it does not have disulfide bonds. Therefore, Fn3 is stable underreducing conditions, unlike antibodies and their fragments (see WO98/56915; WO 01/64942; WO 00/34784). An Fn3 domain can be modified(e.g., using CDRs or hypervariable loops described herein) or varied,e.g., to select domains that bind to IL-13.

Still other exemplary scaffold domains include: T-cell receptors; MHCproteins; extracellular domains (e.g., fibronectin Type III repeats, EGFrepeats); protease inhibitors (e.g., Kunitz domains, ecotin, BPTI, andso forth); TPR repeats; trifoil structures; zinc finger domains;DNA-binding proteins; particularly monomeric DNA binding proteins; RNAbinding proteins; enzymes, e.g., proteases (particularly inactivatedproteases), RNase; chaperones, e.g., thioredoxin, and heat shockproteins; and intracellular signaling domains (such as SH2 and SH3domains). US 20040009530 describes examples of some alternativescaffolds.

Examples of small scaffold domains include: Kunitz domains (58 aminoacids, 3 disulfide bonds), Cucurbida maxima trypsin inhibitor domains(31 amino acids, 3 disulfide bonds), domains related to guanylin (14amino acids, 2 disulfide bonds), domains related to heat-stableenterotoxin IA from gram negative bacteria (18 amino acids, 3 disulfidebonds), EGF domains (50 amino acids, 3 disulfide bonds), kringle domains(60 amino acids, 3 disulfide bonds), fungal carbohydrate-binding domains(35 amino acids, 2 disulfide bonds), endothelin domains (18 amino acids,2 disulfide bonds), and Streptococcal G IgG-binding domain (35 aminoacids, no disulfide bonds). Examples of small intracellular scaffolddomains include SH2, SH3, and EVH domains. Generally, any modulardomain, intracellular or extracellular, can be used.

Exemplary criteria for evaluating a scaffold domain can include: (1)amino acid sequence, (2) sequences of several homologous domains, (3)3-dimensional structure, and/or (4) stability data over a range of pH,temperature, salinity, organic solvent, oxidant concentration. In oneembodiment, the scaffold domain is a small, stable protein domains,e.g., a protein of less than 100, 70, 50, 40 or 30 amino acids. Thedomain may include one or more disulfide bonds or may chelate a metal,e.g., zinc.

Still other binding agents are based on peptides, e.g., proteins with anamino acid sequence that are less than 30, 25, 24, 20, 18, 15, or 12amino acids. Peptides can be incorporated in a larger protein, buttypically a region that can independently bind to IL-13, e.g., to anepitope described herein. Peptides can be identified by phage display.See, e.g., US 20040071705.

An IL-13 binding agent may include non-peptide linkages and otherchemical modification. For example, part or all of the binding agent maybe synthesized as a peptidomimetic, e.g., a peptoid (see, e.g., Simon etal. (1992) Proc. Natl. Acad. Sci. USA 89:9367-71 and Horwell (1995)Trends Biotechnol. 13:132-4). A binding agent may include one or more(e.g., all) non-hydrolyzable bonds. Many non-hydrolyzable peptide bondsare known in the art, along with procedures for synthesis of peptidescontaining such bonds. Exemplary non-hydrolyzable bonds include—[CH₂NH]— reduced amide peptide bonds, —[COCH₂]— ketomethylene peptidebonds, —[CH(CN)NH]— (cyanomethylene)amino peptide bonds, —[CH₂CH(OH)]—hydroxyethylene peptide bonds, —[CH₂O]— peptide bonds, and —[CH₂S]—thiomethylene peptide bonds (see e.g., U.S. Pat. No. 6,172,043).

Pharmaceutical Compositions

The IL-13 binding agents, e.g. antibody molecules that bind to IL-13(such as those described herein) can be used in vitro, ex vivo, or invivo. They can be incorporated into a pharmaceutical composition, e.g.,by combining the IL-13 binding agent with a pharmaceutically acceptablecarrier. Such a composition may contain, in addition to the IL-13binding agent and carrier, various diluents, fillers, salts, buffers,stabilizers, solubilizers, and other materials well known in the art.Pharmaceutically acceptable materials is generally a nontoxic materialthat does not interfere with the effectiveness of the biologicalactivity of an IL-13 binding agent. The characteristics of the carriercan depend on the route of administration.

The pharmaceutical composition described herein may also contain otherfactors, such as, but not limited to, other anti-cytokine antibodymolecules or other anti-inflammatory agents as described in more detailbelow. Such additional factors and/or agents may be included in thepharmaceutical composition to produce a synergistic effect with an IL-13binding agent, e.g., anti-IL-13 antibody molecule, described herein. Forexample, in the treatment of allergic asthma, a pharmaceuticalcomposition described herein may include anti-IL-4 antibody molecules ordrugs known to reduce an allergic response.

The pharmaceutical composition described herein may be in the form of aliposome in which an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, such as one described herein is combined, in addition to otherpharmaceutically acceptable carriers, with amphipathic agents such aslipids that exist in aggregated form as micelles, insoluble monolayers,liquid crystals, or lamellar layers while in aqueous solution. Suitablelipids for liposomal formulation include, without limitation,monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,saponin, bile acids, and the like. Exemplary methods for preparing suchliposomal formulations include methods described in U.S. Pat. Nos.4,235,871; 4,501,728; 4,837,028; and 4,737,323.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, e.g.,amelioration of symptoms of, healing of, or increase in rate of healingof such conditions. When applied to an individual active ingredient,administered alone, the term refers to that ingredient alone. Whenapplied to a combination, the term refers to combined amounts of theactive ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously.

In practicing the method of treatment or use, a therapeuticallyeffective amount of IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, e.g., an antibody molecule that binds to IL-13 and interfereswith the formation of a functional IL-13 signaling complex (and, e.g.,neutralizes or inhibits one or more IL-13-associated activities), isadministered to a subject, e.g., mammal (e.g., a human). An IL-13binding agent, e.g., an anti-IL-13 antibody molecule, may beadministered in accordance with a method described herein either aloneas well as in combination with other therapies such as treatmentsemploying cytokines, lymphokines or other hematopoietic factors, cancertherapeutics, or anti-inflammatory agents. When coadministered with oneor more agents, an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, may be administered either simultaneously with the secondagent, or sequentially. If administered sequentially, a physician canselect an appropriate sequence for administering the IL-13 binding agentin combination with other agents.

Administration of an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, used in the pharmaceutical composition can be carried out in avariety of conventional ways, such as oral ingestion, inhalation, orcutaneous, subcutaneous, or intravenous injection. When atherapeutically effective amount of an IL-13 binding agent, e.g., ananti-IL-13 antibody molecule, is administered by intravenous, cutaneousor subcutaneous injection, the binding agent can be prepared as apyrogen-free, parenterally acceptable aqueous solution. The compositionof such parenterally acceptable protein solutions can be adapted in viewfactors such as pH, isotonicity, stability, and the like, e.g., tooptimize the composition for physiological conditions, binding agentstability, and so forth. A pharmaceutical composition for intravenous,cutaneous, or subcutaneous injection can contain, e.g., an isotonicvehicle such as Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, Lactated Ringer'sInjection, or other vehicle as known in the art. The pharmaceuticalcomposition may also contain stabilizers, preservatives, buffers,antioxidants, or other additive.

The amount of an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, in the pharmaceutical composition can depend upon the natureand severity of the condition being treated, and on the nature of priortreatments that the patient has undergone. The pharmaceuticalcomposition can be administered to normal patients or patients who donot show symptoms, e.g., in a prophylactic mode. An attending physicianmay decide the amount of IL-13 binding agent, e.g., an anti-IL-13antibody molecule, with which to treat each individual patient. Forexample, an attending physician can administer low doses of antagonistand observe the patient's response. Larger doses of antagonist may beadministered until the optimal therapeutic effect is obtained for thepatient, and at that point the dosage is not generally increasedfurther. For example, a pharmaceutical may contain between about 0.1 mgto 50 mg antibody per kg body weight, e.g., between about 0.1 mg and 5mg or between about 8 mg and 50 mg antibody per kg body weight. In oneembodiment in which the antibody is delivered subcutaneously at afrequency of no more than twice per month, e.g., every other week ormonthly, the composition includes an amount of about 0.7-3.3, e.g.,1.0-3.0 mg/kg, e.g., about 0.8-1.2, 1.2-2.8, or 2.8-3.3 mg/kg.

The duration of therapy using the pharmaceutical composition may vary,depending on the severity of the disease being treated and the conditionand potential idiosyncratic response of each individual patient. In oneembodiment, the IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, can also be administered via the subcutaneous route, e.g., inthe range of once a week, once every 24, 48, 96 hours, or not morefrequently than such intervals. Exemplary dosages can be in the range of0.1-20 mg/kg, more preferably 1-10 mg/kg. The agent can be administered,e.g., by intravenous infusion at a rate of less than 20, 10, 5, or 1mg/min to reach a dose of about 1 to 50 mg/m² or about 5 to 20 mg/m².

In one embodiment, an administration of a IL-13 binding agent to thepatient includes varying the dosage of the protein, e.g., to reduce orminimize side effects. For example, the subject can be administered afirst dosage, e.g., a dosage less than a therapeutically effectiveamount. In a subsequent interval, e.g., at least 6, 12, 24, or 48 hourslater, the patient can be administered a second dosage, e.g., a dosagethat is at least 25, 50, 75, or 100% greater than the first dosage. Forexample, the second and/or a comparable third, fourth and fifth dosagecan be at least about 70, 80, 90, or 100% of a therapeutically effectiveamount.

Inhalation

A composition that includes an IL-13 binding agent, e.g., an anti-IL-13antibody molecule, can be formulated for inhalation or other mode ofpulmonary delivery. Accordingly, the IL-13 binding agent can beadministered by inhalation to pulmonary tissue. The term “pulmonarytissue” as used herein refers to any tissue of the respiratory tract andincludes both the upper and lower respiratory tract, except whereotherwise indicated. An IL-13 binding agent, e.g., an anti-IL-13antibody molecule, can be administered in combination with one or moreof the existing modalities for treating pulmonary diseases.

In one example the IL-13 binding agent is formulated for a nebulizer. Inone embodiment, the IL-13 binding agent can be stored in a lyophilizedform (e.g., at room temperature) and reconstituted in solution prior toinhalation. It is also possible to formulate the IL-13 binding agent forinhalation using a medical device, e.g., an inhaler. See, e.g., U.S.Pat. No. 6,102,035 (a powder inhaler) and 6,012,454 (a dry powderinhaler). The inhaler can include separate compartments for the IL-13binding agent at a pH suitable for storage and another compartment for aneutralizing buffer and a mechanism for combining the IL-13 bindingagent with a neutralizing buffer immediately prior to atomization. Inone embodiment, the inhaler is a metered dose inhaler.

The three common systems used to deliver drugs locally to the pulmonaryair passages include dry powder inhalers (DPIs), metered dose inhalers(MDIs) and nebulizers. MDIs, the most popular method of inhalationadministration, may be used to deliver medicaments in a solubilized formor as a dispersion. Typically MDIs comprise a Freon or other relativelyhigh vapor pressure propellant that forces aerosolized medication intothe respiratory tract upon activation of the device. Unlike MDIs, DPIsgenerally rely entirely on the inspiratory efforts of the patient tointroduce a medicament in a dry powder form to the lungs. Nebulizersform a medicament aerosol to be inhaled by imparting energy to a liquidsolution. Direct pulmonary delivery of drugs during liquid ventilationor pulmonary lavage using a fluorochemical medium has also beenexplored. These and other methods can be used to deliver an IL-13binding agent, e.g., anti-IL-13 antibody molecule. In one embodiment,the IL-13 binding agent is associated with a polymer, e.g., a polymerthat stabilizes or increases half-life of the compound.

For example, for administration by inhalation, an IL-13 binding agent,e.g., an anti-IL-13 antibody molecule, is delivered in the form of anaerosol spray from pressured container or dispenser which contains asuitable propellant or a nebulizer. The IL-13 binding agent may be inthe form of a dry particle or as a liquid. Particles that include theIL-13 binding agent can be prepared, e.g., by spray drying, by drying anaqueous solution of the IL-13 binding agent, e.g., an anti-IL-13antibody molecule, with a charge neutralizing agent and then creatingparticles from the dried powder or by drying an aqueous solution in anorganic modifier and then creating particles from the dried powder.

The IL-13 binding agent may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges for use in an inhaler or insufflator may beformulated containing a powder mix of an IL-13 binding agent, e.g., ananti-IL-13 antibody molecule, and a suitable powder base such as lactoseor starch, if the particle is a formulated particle. In addition to theformulated or unformulated compound, other materials such as 100% DPPCor other surfactants can be mixed with the IL-13 binding agent topromote the delivery and dispersion of formulated or unformulatedcompound. Methods of preparing dry particles are described, for example,in WO 02/32406.

An IL-13 binding agent, e.g., an anti-IL-13 antibody molecule, can beformulated for aerosol delivery, e.g., as dry aerosol particles, suchthat when administered it can be rapidly absorbed and can produce arapid local or systemic therapeutic result. Administration can betailored to provide detectable activity within 2 minutes, 5 minutes, 1hour, or 3 hours of administration. In some embodiments, the peakactivity can be achieved even more quickly, e.g., within one half houror even within ten minutes. An IL-13 binding agent, e.g., an anti-IL-13antibody molecule, can be formulated for longer biological half-life(e.g., by association with a polymer such as PEG) for use as analternative to other modes of administration, e.g., such that the IL-13binding agent enters circulation from the lung and is distributed toother organs or to a particular target organ.

In one embodiment, the IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, is delivered in an amount such that at least 5% of the mass ofthe polypeptide is delivered to the lower respiratory tract or the deeplung. Deep lung has an extremely rich capillary network. The respiratorymembrane separating capillary lumen from the alveolar air space is verythin (≦6 Tm) and extremely permeable. In addition, the liquid layerlining the alveolar surface is rich in lung surfactants. In otherembodiments, at least 2%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or80% of the composition of an IL-13 binding agent, e.g., an anti-IL-13antibody molecule, is delivered to the lower respiratory tract or to thedeep lung. Delivery to either or both of these tissues results inefficient absorption of the IL-13 binding agent and highbioavailability. In one embodiment, the IL-13 binding agent is providedin a metered dose using, e.g., an inhaler or nebulizer. For example, theIL-13 binding agent is delivered in a dosage unit form of at least about0.02, 0.1, 0.5, 1, 1.5, 2, 5, 10, 20, 40, or 50 mg/puff or more. Thepercent bioavailability can be calculated as follows: the percentbioavailability=(AUC_(non-invasive)/AUC_(i.v. or s.c.))×(dose_(i.v. or s.c)./dose_(non-invasive))×100.

Although not necessary, delivery enhancers such as surfactants can beused to further enhance pulmonary delivery. A “surfactant” as usedherein refers to a IL-13 binding agent having a hydrophilic andlipophilic moiety, which promotes absorption of a drug by interactingwith an interface between two immiscible phases. Surfactants are usefulin the dry particles for several reasons, e.g., reduction of particleagglomeration, reduction of macrophage phagocytosis, etc. When coupledwith lung surfactant, a more efficient absorption of the IL-13 bindingagent can be achieved because surfactants, such as DPPC, will greatlyfacilitate diffusion of the compound. Surfactants are well known in theart and include but are not limited to phosphoglycerides, e.g.,phosphatidylcholines, L-alpha-phosphatidylcholine dipalmitoyl (DPPC) anddiphosphatidyl glycerol (DPPG); hexadecanol; fatty acids; polyethyleneglycol (PEG); polyoxyethylene-9-; auryl ether; palmitic acid; oleicacid; sorbitan trioleate (Span 85); glycocholate; surfactin; poloxomer;sorbitan fatty acid ester; sorbitan trioleate; tyloxapol; andphospholipids.

Stabilization

In one embodiment, an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, is physically associated with a moiety that improves itsstabilization and/or retention in circulation, e.g., in blood, serum,lymph, bronchopulmonary lavage, or other tissues, e.g., by at least 1.5,2, 5, 10, or 50 fold.

For example, an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, can be associated with a polymer, e.g., a substantiallynon-antigenic polymers, such as polyalkylene oxides or polyethyleneoxides. Suitable polymers will vary substantially by weight. Polymershaving molecular number average weights ranging from about 200 to about35,000 (or about 1,000 to about 15,000, and 2,000 to about 12,500) canbe used.

For example, an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, can be conjugated to a water soluble polymer, e.g.,hydrophilic polyvinyl polymers, e.g. polyvinylalcohol andpolyvinylpyrrolidone. A non-limiting list of such polymers includespolyalkylene oxide homopolymers such as polyethylene glycol (PEG) orpolypropylene glycols, polyoxyethylenated polyols, copolymers thereofand block copolymers thereof, provided that the water solubility of theblock copolymers is maintained. Additional useful polymers includepolyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and blockcopolymers of polyoxyethylene and polyoxypropylene (Pluronics);polymethacrylates; carbomers; branched or unbranched polysaccharideswhich comprise the saccharide monomers D-mannose, D- and L-galactose,fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid,D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid, oralginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminicacid including homopolysaccharides and heteropolysaccharides such aslactose, amylopectin, starch, hydroxyethyl starch, amylose, dextransulfate, dextran, dextrins, glycogen, or the polysaccharide subunit ofacid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugaralcohols such as polysorbitol and polymannitol; heparin or heparan.

The conjugates of an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, and a polymer can be separated from the unreacted startingmaterials, e.g., by gel filtration or ion exchange chromatography, e.g.,HPLC. Heterologous species of the conjugates are purified from oneanother in the same fashion. Resolution of different species (e.g.containing one or two PEG residues) is also possible due to thedifference in the ionic properties of the unreacted amino acids. See,e.g., WO 96/34015.

Use of IL-13 Binding Agents to Modulate One or More IL-13-AssociatedActivities In Vivo

In yet another aspect, the invention features a method for modulating(e.g., decreasing, neutralizing and/or inhibiting) one or moreassociated activities of IL-13 in vivo by administering an IL-13 bindingagent, e.g., an anti-IL-13 antibody molecule, described herein in anamount sufficient to inhibit its activity. An IL-13 binding agent canalso be administered to subjects for whom inhibition of anIL-13-mediated inflammatory response is required. These conditionsinclude, e.g., airway inflammation, asthma, fibrosis, eosinophilia andincreased mucus production.

The efficacy of an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, described herein can be evaluated, e.g., by evaluating abilityof the antagonist to modulate airway inflammation in cynomolgus monkeysexposed to an Ascaris suum allergen. An IL-13 binding agent,particularly one that inhibits at least one IL-13 activity, can be usedto neutralize or inhibit one or more IL-13-associated activities, e.g.,to reduce IL-13 mediated inflammation in vivo, e.g., for treating orpreventing IL-13-associated pathologies, including asthma and/or itsassociated symptoms.

In one embodiment, an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, e.g., pharmaceutical compositions thereof, is administered incombination therapy, i.e., combined with other agents, e.g., therapeuticagents, that are useful for treating pathological conditions ordisorders, such as allergic and inflammatory disorders. The term “incombination” in this context means that the agents are givensubstantially contemporaneously, either simultaneously or sequentially.If given sequentially, at the onset of administration of the secondcompound, the first of the two compounds is preferably still detectableat effective concentrations at the site of treatment.

For example, the combination therapy can include one or more IL-13binding agents, e.g., anti-IL-13 antibodies and fragments thereof, e.g.,that bind to IL-13 and interfere with the formation of a functionalIL-13 signaling complex, coformulated with, and/or coadministered with,one or more additional therapeutic agents, e.g., one or more cytokineand growth factor inhibitors, immunosuppressants, anti-inflammatoryagents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic orcytostatic agents, as described in more detail below. Furthermore, oneor more an IL-13 binding agent, e.g., an anti-IL-13 antibody molecule,may be used in combination with two or more of the therapeutic agentsdescribed herein. Such combination therapies may advantageously utilizelower dosages of the administered therapeutic agents, thus avoidingpossible toxicities or complications associated with the variousmonotherapies. Moreover, the therapeutic agents disclosed herein act onpathways that differ from the IL-13/IL-13-receptor pathway, and thus areexpected to enhance and/or synergize with the effects of the IL-13binding agents.

Therapeutic agents that interfere with different triggers of asthma orairway inflammation, e.g., therapeutic agents used in the treatment ofallergy, upper respiratory infections, or ear infections, may be used incombination with an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule. In one embodiment, one or more IL-13 binding agents, e.g.,anti-IL-13 antibodies and fragments thereof, may be coformulated with,and/or coadministered with, one or more additional agents, such as othercytokine or growth factor antagonists (e.g., soluble receptors, peptideinhibitors, small molecules, adhesins), antibody molecules that bind toother targets (e.g., antibodies that bind to other cytokines or growthfactors, their receptors, or other cell surface molecules), andanti-inflammatory cytokines or agonists thereof. Nonlimiting examples ofthe agents that can be used in combination with IL-13 binding agents,e.g., anti-IL-13 antibodies and fragments thereof, include, but are notlimited to, inhaled steroids; beta-agonists, e.g., short-acting orlong-acting beta-agonists; antagonists of leukotrienes or leukotrienereceptors; combination drugs such as ADVAIR®; IgE inhibitors, e.g.,anti-IgE antibodies (e.g., XOLAIR®); phosphodiesterase inhibitors (e.g.,PDE4 inhibitors); xanthines; anticholinergic drugs; mastcell-stabilizing agents such as cromolyn; IL-4 inhibitors; IL-5inhibitors; eotaxin/CCR3 inhibitors; and antihistamines.

In other embodiments, one or more IL-13 binding agents, e.g., anti-IL-13antibody molecules, can be coformulated with, and/or coadministeredwith, one or more anti-inflammatory drugs, immunosuppressants, ormetabolic or enzymatic inhibitors. Examples of the drugs or inhibitorsthat can be used in combination with the IL-13 binding agents, e.g.,anti-IL-13 antibodies and fragments thereof, include, but are notlimited to, one or more of: Additional examples of therapeutic agentsthat can be coadministered and/or coformulated with one or moreanti-IL-13 antibodies or fragments thereof include one or more of: TNFantagonists (e.g., a soluble fragment of a TNF receptor, e.g., p55 orp75 human TNF receptor or derivatives thereof, e.g., 75 kd TNFR-IgG (75kD TNF receptor-IgG fusion protein, ENBREL™)); TNF enzyme antagonists,e.g., TNFα converting enzyme (TACE) inhibitors; muscarinic receptorantagonists; TGF-υ antagonists; interferon gamma; perfenidone;chemotherapeutic agents, e.g., methotrexate, leflunomide, or a sirolimus(rapamycin) or an analog thereof, e.g., CCI-779; COX2 and cPLA2inhibitors; NSAIDs; immunomodulators; p38 inhibitors, TPL-2, Mk-2 andNFPB inhibitors, among others.

Vaccine Formulations

In another aspect, the invention features a method of modifying animmune response associated with immunization. An IL-13 binding agent(e.g., an anti-IL-13 antibody molecule), can be used to increase theefficacy of immunization by inhibiting IL-13 activity. IL-13 bindingagents can be administered before, during, or after delivery of animmunogen, e.g., administration of a vaccine. In one embodiment, theimmunity raised by the vaccination is a cellular immunity, e.g., animmunity against cancer cells or virus infected, e.g., retrovirusinfected, e.g., HIV infected, cells. In one embodiment, the vaccineformulation contains one or more IL-13 binding agents and an antigen,e.g., an immunogen. In another embodiment, the IL-13 binding agent andthe immunogen are administered separately, e.g., within one hour, threehours, one day, or two days of each other. The IL-13 binding agent canbe one that neutralizes or inhibits one or more IL-13 activities.

Inhibition of IL-13 can improve the efficacy of, e.g., cellularvaccines, e.g., vaccines against diseases such as cancer and viralinfection, e.g., retroviral infection, e.g., HIV infection. Induction ofCD8⁺ cytotoxic T lymphocytes (CTL) by vaccines is down modulated by CD4⁺T cells, likely through the cytokine IL-13. Inhibition of IL-13 has beenshown to enhance vaccine induction of CTL response (Ahlers et al. (2002)Proc. Natl. Acad. Sci. USA 99:13020-10325). An IL-13 binding agent,e.g., an anti-IL-13 antibody molecule, an antibody described herein, canbe used in conjunction with a vaccine to increase vaccine efficacy.Cancer and viral infection (such as retroviral (e.g., HIV) infection)are exemplary disorders against which a cellular vaccine response can beeffective. Vaccine efficacy is enhanced by blocking IL-13 signaling atthe time of vaccination (Ahlers et al. (2002) Proc. Nat. Acad. Sci. USA99:13020-25). A vaccine formulation may be administered to a subject inthe form of a pharmaceutical or therapeutic composition.

Methods for Diagnosing, Prognosing, and Monitoring Disorders

IL-13 binding agents can be used in vitro and in vivo as diagnosticagents. One exemplary method includes: (i) administering the IL-13binding agent (e.g., an IL-13 antibody molecule) to a subject; and (ii)detecting the IL-13 binding agent in the subject. The detecting caninclude determining location of the IL-13 binding agent in the subject.Another exemplary method includes contacting an IL-13 binding agent to asample, e.g., a sample from a subject. The presence or absence of IL-13or the level of IL-13 (either qualitative or quantitative) in the samplecan be determined.

In another aspect, the present invention provides a diagnostic methodfor detecting the presence of a IL-13, in vitro (e.g., a biologicalsample, such as tissue, biopsy) or in vivo (e.g., in vivo imaging in asubject).

The method includes: (i) contacting a sample with IL-13 binding agent;and (ii) detecting formation of a complex between the IL-13 bindingagent and the sample. The method can also include contacting a referencesample (e.g., a control sample) with the binding agent, and determiningthe extent of formation of the complex between the binding agent an thesample relative to the same for the reference sample. A change, e.g., astatistically significant change, in the formation of the complex in thesample or subject relative to the control sample or subject can beindicative of the presence of IL-13 in the sample.

Another method includes: (i) administering the IL-13 binding agent to asubject; and (ii) detecting formation of a complex between the IL-13binding agent and the subject. The detecting can include determininglocation or time of formation of the complex.

The IL-13 binding agent can be directly or indirectly labeled with adetectable substance to facilitate detection of the bound or unboundprotein. Suitable detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials andradioactive materials.

Complex formation between the IL-13 binding agent and IL-13 can bedetected by measuring or visualizing either the binding agent bound tothe IL-13 or unbound binding agent. Conventional detection assays can beused, e.g., an enzyme-linked immunosorbent assays (ELISA), aradioimmunoassay (RIA) or tissue immunohistochemistry. Further tolabeling the IL-13 binding agent, the presence of IL-13 can be assayedin a sample by a competition immunoassay utilizing standards labeledwith a detectable substance and an unlabeled IL-13 binding agent. In oneexample of this assay, the biological sample, the labeled standards andthe IL-13 binding agent are combined and the amount of labeled standardbound to the unlabeled binding agent is determined. The amount of IL-13in the sample is inversely proportional to the amount of labeledstandard bound to the IL-13 binding agent.

Methods for Diagnosing, Prognosing, and/or Monitoring Asthma

The binding agents described herein can be used, e.g., in methods fordiagnosing, prognosing, and monitoring the progress of asthma bymeasuring the level of IL-13 in a biological sample. In addition, thisdiscovery enables the identification of new inhibitors of IL-13signaling, which will also be useful in the treatment of asthma.

Such methods for diagnosing allergic and nonallergic asthma can includedetecting an alteration (e.g., a decrease or increase) of IL-13 in abiological sample, e.g., serum, plasma, bronchoalveolar lavage fluid,sputum, etc. “Diagnostic” or “diagnosing” means identifying the presenceor absence of a pathologic condition. Diagnostic methods involvedetecting the presence of IL-13 by determining a test amount of IL-13polypeptide in a biological sample, e.g., in bronchoalveolar lavagefluid, from a subject (human or nonhuman mammal), and comparing the testamount with a normal amount or range (i.e., an amount or range from anindividual(s) known not to suffer from asthma) for the IL-13polypeptide. While a particular diagnostic method may not provide adefinitive diagnosis of asthma, it suffices if the method provides apositive indication that aids in diagnosis.

Methods for prognosing asthma and/or atopic disorders can includedetecting upregulation of IL-13, at the mRNA or protein level.“Prognostic” or “prognosing” means predicting the probable developmentand/or severity of a pathologic condition. Prognostic methods involvedetermining the test amount of IL-13 in a biological sample from asubject, and comparing the test amount to a prognostic amount or range(i.e., an amount or range from individuals with varying severities ofasthma) for IL-13. Various amounts of the IL-13 in a test sample areconsistent with certain prognoses for asthma. The detection of an amountof IL-13 at a particular prognostic level provides a prognosis for thesubject.

The present application also provides methods for monitoring the courseof asthma by detecting the upregulation of IL-13. Monitoring methodsinvolve determining the test amounts of IL-13 in biological samplestaken from a subject at a first and second time, and comparing theamounts. A change in amount of IL-13 between the first and second timecan indicate a change in the course of asthma and/or atopic disorder,with a decrease in amount indicating remission of asthma, and anincrease in amount indicating progression of asthma and/or atopicdisorder. Such monitoring assays are also useful for evaluating theefficacy of a particular therapeutic intervention (e.g., diseaseattenuation and/or reversal) in patients being treated for an IL-13associated disorder.

Fluorophore- and chromophore-labeled binding agents can be prepared. Thefluorescent moieties can be selected to have substantial absorption atwavelengths above 310 nm, and preferably above 400 nm. A variety ofsuitable fluorescers and chromophores are described by Stryer (1968)Science, 162:526 and Brand, L. et al. (1972) Annual Review ofBiochemistry, 41:843-868. The binding agents can be labeled withfluorescent chromophore groups by conventional procedures such as thosedisclosed in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110. Onegroup of fluorescers having a number of the desirable propertiesdescribed above is the xanthene dyes, which include the fluoresceins andrhodamines. Another group of fluorescent compounds are thenaphthylamines. Once labeled with a fluorophore or chromophore, thebinding agent can be used to detect the presence or localization of theIL-13 in a sample, e.g., using fluorescent microscopy (such as confocalor deconvolution microscopy).

Histological Analysis. Immunohistochemistry can be performed using thebinding agents described herein. For example, in the case of anantibody, the antibody can synthesized with a label (such as apurification or epitope tag), or can be detectably labeled, e.g., byconjugating a label or label-binding group. For example, a chelator canbe attached to the antibody. The antibody is then contacted to ahistological preparation, e.g., a fixed section of tissue that is on amicroscope slide. After an incubation for binding, the preparation iswashed to remove unbound antibody. The preparation is then analyzed,e.g., using microscopy, to identify if the antibody bound to thepreparation. The antibody (or other polypeptide or peptide) can beunlabeled at the time of binding. After binding and washing, theantibody is labeled in order to render it detectable.

Protein Arrays. An IL-13 binding agent (e.g., a protein that is an IL-13binding agent) can also be immobilized on a protein array. The proteinarray can be used as a diagnostic tool, e.g., to screen medical samples(such as isolated cells, blood, sera, biopsies, and the like). Theprotein array can also include other binding agents, e.g., ones thatbind to IL-13 or to other target molecules.

Methods of producing protein arrays are described, e.g., in De Wildt etal. (2000) Nat. Biotechnol. 18:989-994; Lueking et al. (1999) Anal.Biochem. 270:103-111; Ge (2000) Nucleic Acids Res. 28, e3, I-VII;MacBeath and Schreiber (2000) Science 289:1760-1763; WO 01/40803 and WO99/51773A1. Polypeptides for the array can be spotted at high speed,e.g., using commercially available robotic apparati, e.g., from GeneticMicroSystems or BioRobotics. The array substrate can be, for example,nitrocellulose, plastic, glass, e.g., surface-modified glass. The arraycan also include a porous matrix, e.g., acrylamide, agarose, or anotherpolymer. For example, the array can be an array of antibodies, e.g., asdescribed in De Wildt, supra. Cells that produce the protein can begrown on a filter in an arrayed format. proteins production is induced,and the expressed protein are immobilized to the filter at the locationof the cell.

A protein array can be contacted with a sample to determine the extentof IL-13 in the sample. If the sample is unlabeled, a sandwich methodcan be used, e.g., using a labeled probe, to detect binding of theIL-13. Information about the extent of binding at each address of thearray can be stored as a profile, e.g., in a computer database. Theprotein array can be produced in replicates and used to compare bindingprofiles, e.g., of different samples.

Flow Cytometry. The IL-13 binding agent can be used to label cells,e.g., cells in a sample (e.g., a patient sample). The binding agent canbe attached (or attachable) to a fluorescent compound. The cells canthen be analyzed by flow cytometry and/or sorted using fluorescentactivated cell sorted (e.g., using a sorter available from BectonDickinson Immunocytometry Systems, San Jose Calif.; see also U.S. Pat.Nos. 5,627,037; 5,030,002; and 5,137,809). As cells pass through thesorter, a laser beam excites the fluorescent compound while a detectorcounts cells that pass through and determines whether a fluorescentcompound is attached to the cell by detecting fluorescence. The amountof label bound to each cell can be quantified and analyzed tocharacterize the sample. The sorter can also deflect the cell andseparate cells bound by the binding agent from those cells not bound bythe binding agent. The separated cells can be cultured and/orcharacterized.

In vivo Imaging. In still another embodiment, the invention provides amethod for detecting the presence of a IL-13 within a subject in vivo.The method includes (i) administering to a subject (e.g., a patienthaving an IL-13 associated disorder) an anti-IL-13 antibody, conjugatedto a detectable marker; (ii) exposing the subject to a means fordetecting the detectable marker. For example, the subject is imaged,e.g., by

NMR or other tomographic means.

Examples of labels useful for diagnostic imaging include radiolabelssuch as ¹³¹I, ¹¹¹In, ¹²³I, ^(99m)Tc, ³²P, ³³P, ¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh,fluorescent labels such as fluorescein and rhodamine, nuclear magneticresonance active labels, positron emitting isotopes detectable by apositron emission tomography (“PET”) scanner, chemiluminescers such asluciferin, and enzymatic markers such as peroxidase or phosphatase.Short-range radiation emitters, such as isotopes detectable byshort-range detector probes can also be employed. The binding agent canbe labeled with such reagents using known techniques. For example, seeWensel and Meares (1983) Radioimmunoimaging and Radioimmunotherapy,Elsevier, N.Y. for techniques relating to the radiolabeling ofantibodies and Colcher et al. (1986) Meth. Enzymol. 121: 802-816. Aradiolabeled binding agent can also be used for in vitro diagnostictests. The specific activity of a isotopically-labeled binding agentdepends upon the half-life, the isotopic purity of the radioactivelabel, and how the label is incorporated into the antibody. Proceduresfor labeling polypeptides with the radioactive isotopes (such as ¹⁴C,³H, ³⁵S, ¹²⁵I, ^(99m)Tc, ³²P, ³³P, and ¹³¹I) are generally known. See,e.g., U.S. Pat. No. 4,302,438; Goding, J. W. (Monoclonal antibodies:principles and practice: production and application of monoclonalantibodies in cell biology, biochemistry, and immunology 2nd ed. London;Orlando: Academic Press, 1986. pp 124-126) and the references citedtherein; and A. R. Bradwell et al., “Developments in Antibody Imaging”,Monoclonal Antibodies for Cancer Detection and Therapy, R. W. Baldwin etal., (eds.), pp 65-85 (Academic Press 1985).

IL-13 binding agents described herein can be conjugated to MagneticResonance Imaging (MRI) contrast agents. Some MRI techniques aresummarized in EP-A-0 502 814. Generally, the differences in relaxationtime constants T1 and T2 of water protons in different environments isused to generate an image. However, these differences can beinsufficient to provide sharp high resolution images. The differences inthese relaxation time constants can be enhanced by contrast agents.Examples of such contrast agents include a number of magnetic agentsparamagnetic agents (which primarily alter T1) and ferromagnetic orsuperparamagnetic (which primarily alter T2 response). Chelates (e.g.,EDTA, DTPA and NTA chelates) can be used to attach (and reduce toxicity)of some paramagnetic substances (e.g., Fe³⁺, Mn²⁺, Gd³⁺). Other agentscan be in the form of particles, e.g., less than 10 μm to about 10 nm indiameter) and having ferromagnetic, antiferromagnetic, orsuperparamagnetic properties. The IL-13 binding agents can also belabeled with an indicating group containing the NMR active ¹⁹F atom, asdescribed by Pykett (1982) Scientific American, 246:78-88 to locate andimage IL-13 distribution.

Also within the scope described herein are kits comprising an IL-13binding agent and instructions for diagnostic use, e.g., the use of theIL-13 binding agent (e.g., an antibody molecule or other polypeptide orpeptide) to detect IL-13, in vitro, e.g., in a sample, e.g., a biopsy orcells from a patient having an IL-13 associated disorder, or in vivo,e.g., by imaging a subject. The kit can further contain a least oneadditional reagent, such as a label or additional diagnostic agent. Forin vivo use the binding agent can be formulated as a pharmaceuticalcomposition.

Kits

An IL-13 binding agent, e.g., an anti-IL-13 antibody molecule, can beprovided in a kit, e.g., as a component of a kit. For example, the kitincludes (a) an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, and, optionally (b) informational material. The informationalmaterial can be descriptive, instructional, marketing or other materialthat relates to a method, e.g., a method described herein. Theinformational material of the kits is not limited in its form. In oneembodiment, the informational material can include information aboutproduction of the compound, molecular weight of the compound,concentration, date of expiration, batch or production site information,and so forth. In one embodiment, the informational material relates tousing the IL-13 binding agent to treat, prevent, diagnose, prognose, ormonitor a disorder described herein.

In one embodiment, the informational material can include instructionsto administer an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, in a suitable manner to perform the methods described herein,e.g., in a suitable dose, dosage form, or mode of administration (e.g.,a dose, dosage form, or mode of administration described herein). Inanother embodiment, the informational material can include instructionsto administer an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule, to a suitable subject, e.g., a human, e.g., a human having, orat risk for, allergic asthma, non-allergic asthma, or an IL-13 mediateddisorder, e.g., an allergic and/or inflammatory disorder, or HTLV-1infection. IL-13 production has been correlated with HTLV-1 infection(Chung et al., (2003) Blood 102: 4130-36).

For example, the material can include instructions to administer anIL-13 binding agent, e.g., an anti-IL-13 antibody molecule, to apatient, a patient with or at risk for allergic asthma, non-allergicasthma, or an IL-13 mediated disorder, e.g., an allergic and/orinflammatory disorder, or HTLV-1 infection.

The kit can include one or more containers for the compositioncontaining an IL-13 binding agent, e.g., an anti-IL-13 antibodymolecule. In some embodiments, the kit contains separate containers,dividers or compartments for the composition and informational material.For example, the composition can be contained in a bottle, vial, orsyringe, and the informational material can be contained in a plasticsleeve or packet. In other embodiments, the separate elements of the kitare contained within a single, undivided container. For example, thecomposition is contained in a bottle, vial or syringe that has attachedthereto the informational material in the form of a label. In someembodiments, the kit includes a plurality (e.g., a pack) of individualcontainers, each containing one or more unit dosage forms (e.g., adosage form described herein) of an IL-13 binding agent, e.g.,anti-IL-13 antibody molecule. For example, the kit includes a pluralityof syringes, ampules, foil packets, atomizers or inhalation devices,each containing a single unit dose of an IL-13 binding agent, e.g., ananti-IL-13 antibody molecule, or multiple unit doses.

The kit optionally includes a device suitable for administration of thecomposition, e.g., a syringe, inhalant, pipette, forceps, measuredspoon, dropper (e.g., eye dropper), swab (e.g., a cotton swab or woodenswab), or any such delivery device. In a preferred embodiment, thedevice is an implantable device that dispenses metered doses of thebinding agent.

The Examples that follow are set forth to aid in the understanding ofthe inventions but are not intended to, and should not be construed to,limit its scope in any way.

EXAMPLES Example 1 (a) Cloning of NHP-IL-13 and Homology to Human IL-13

The cynomolgus monkey IL-13 (NHP IL-13) was cloned using hybridizationprobes. A comparison of the cynomolgus monkey IL-13 amino acid sequenceto that of human IL-13 is shown in FIG. 1A. There is 94% amino acididentity between the two sequences, due to 8 amino acid differences. Oneof these differences, R130Q, represents a common human polymorphismpreferentially expressed in asthmatic subjects (Heinzmann et al. (2000)Hum. Mol. Genet. 9:549-559).

(b) Binding of NHP-IL-13 to Human IL13Rα2

Human IL-13 binds with high affinity to the alpha2 form of IL-13receptor (IL13Rα2). A soluble form of this receptor was expressed with ahuman IgG1 Fc tail (sIL13Rα2-Fc). By binding to IL-13 and sequesteringthe cytokine from the cell surface IL13Rα1-IL4R signaling complex,sIL13Rα2-Fc can act as a potent inhibitor of human IL-13 bioactivity.sIL13Rα2-Fc was shown to bind to NHP-IL-13 produced by CHO cells or E.coli.

(c) Bioactivity of NHP-IL-13 on Human Monocytes

(i) CD23 expression on human monocytes. cDNA encoding cynomolgus monkeyIL-13 was expressed in E. coli and refolded to maintain bioactivity.Reactivity of human cells to cynomolgus IL-13 was demonstrated using abioassay in which normal peripheral blood mononuclear cells from healthydonors were treated with IL-13 overnight at 37° C. This inducedup-regulation of CD23 expression on the surface of monocytes. Resultsshowed that cynomolgus IL-13 had bioactivity on primary human monocytes.

(ii) STAT6 phosphorylation on HT-29 cells. The human HT-29 epithelialcell line responds to IL-13 by undergoing STAT6 phosphorylation, aconsequence of signal transduction through the IL-13 receptor. To assaythe ability of recombinant NHP-IL-13 to induce STAT6 phosphorylation,HT-29 cells were challenged with the NHP-IL-13 for 30 minutes at 37° C.,then fixed, permeabilized, and stained with fluorescent antibody tophospho-STAT6. Results showed that cynomolgus IL-13 efficiently inducedSTAT6 phosphorylation in this human cell line.

(d) Generation of Antibodies that Bind to NHP-IL-13

Mice or other appropriate animals may be immunized and boosted withcynomolgus IL-13, e.g., using one or more of the following methods. Onemethod for immunization may be combined with either the same ordifferent method for boosting:

(i) Immunization with cynomolgus IL-13 protein expressed in E. coli,purified from inclusion bodies, and refolded to preserve biologicalactivity. For immunization, the protein is emulsified with completeFreund's adjuvant (CFA), and mice are immunized according to standardprotocols. For boosting, the same protein is emulsified with incompleteFreund's adjuvant (IFA).

(ii) Immunization with peptides spanning the entire sequence of maturecynomolgus IL-13. Each peptide contains at least one amino acid that isunique to cynomolgus IL-13 and not present in the human protein. SeeFIG. 1B. Where the peptide has a C-terminal residue other than cysteine,a cysteine is added for conjugation to a carrier protein. The peptidesare conjugated to an immunogenic carrier protein such as KLH, and usedto immunize mice according to standard protocols. For immunization, theprotein is emulsified with complete Freund's adjuvant (CFA), and miceare immunized according to standard protocols. For boosting, the sameprotein is emulsified with incomplete Freund's adjuvant (IFA).

(iii) Immunization with NHP-IL-13-encoding cDNA expressed. The cDNAencoding NHP-IL-13, including leader sequence, is cloned into anappropriate vector. This DNA is coated onto gold beads which areinjected intradermally by gene gun.

(iv) The protein or peptides can be used as a target for screening aprotein library, e.g., a phage or ribosome display library. For example,the library can display varied immunoglobulin molecules, e.g., Fab's,scFv's, or Fd's.

(e) Selection of Antibody Clones Cross-Reactive with NHP and Optionallya Human IL-13, e.g., a Native Human IL-13

Primary Screen

The primary screen for antibodies was selection for binding torecombinant NHP-IL-13 by ELISA. In this ELISA, wells are coated withrecombinant NHP IL-13. The immune serum was added in serial dilutionsand incubated for one hour at room temperature. Wells were washed withPBS containing 0.05% TWEEN®-20 (PBS-Tween). Bound antibody was detectedusing horseradish peroxidase (HRP)-labeled anti-mouse IgG andtetramethylbenzidene (TMB) substrate. Absorbance was read at 450 nm.Typically, all immunized mice generated high titers of antibody toNHP-IL-13.

Secondary Screen

The secondary screen was selection for inhibition of binding ofrecombinant NHP-IL-13 to sIL-13Rα1-Fc by ELISA. Wells were coated withsoluble IL-13Rα1-Fc, to which FLAG-tagged NHP-IL-13 could bind. Thisbinding was detected with anti-FLAG antibody conjugated to HRP.Hydrolysis of TMB substrate was read as absorbance at 450 nm. In theassay, the FLAG-tagged NHP-IL-13 was added together with increasingconcentrations of immune serum. If the immune serum contained antibodythat bound to NHP-IL-13 and prevented its binding to the sIL13Rα1-Fccoating the wells, the ELISA signal was decreased. All immunized miceproduced antibody that competed with sIL13Rα1-Fc binding to NHP-IL-13,but the titers varied from mouse to mouse. Spleens were selected forfusion from animals whose serum showed inhibited sIL13Rα1-Fc binding toNHP-IL-13 at the highest dilution.

Tertiary Screen

The tertiary screen tested for inhibition of NHP-IL-13 bioactivity.Several bioassays were available to be used, including the TF-1proliferation assay, the monocyte CD23 expression assay, and the HT-29cell STAT6 phosphorylation assay. Immune sera were tested for inhibitionof NHP-IL-13-mediated STAT6 phosphorylation. The HT-29 human epithelialcell line was challenged for 30 minutes at 37° C. with recombinantNHP-IL-13 in the presence or absence of the indicated concentration ofmouse immune serum. Cells were then fixed, permeabilized, and stainedwith ALEXA3 Fluor 488-conjugated mAb to phospho-STAT6 (Pharmingen). Thepercentage of cells responding to IL-13 by undergoing STAT6phosphorylation was determined by flow cytometry. Spleens of mice withthe most potent neutralization activity, determined as the strongestinhibition of NHP-IL-13 bioactivity at a high serum dilution, wereselected for generation of hybridomas.

Quaternary Screen

A crude preparation containing human IL-13 was generated from humanumbilical cord blood mononuclear cells (BioWhittaker/Cambrex). The cellswere cultured in a 37° C. incubator at 5% CO₂, in RPMI media containing10% heat-inactivated FCS, 50 U/ml penicillin, 50 mg/ml streptomycin, and2 mM L-glutamine. Cells were stimulated for 3 days with the mitogenPHA-P (Sigma), and skewed toward Th2 with recombinant human IL-4 (R&DSystems) and anti-human IL-12. The Th2 cells were expanded for one weekwith IL-2, then activated to produce cytokine by treatment with phorbol12-myristate 13-acetate (PMA) and ionomycin for three days. Thesupernatant was collected and dialyzed to remove PMA and ionomycin. Todeplete GM-CSF and IL-4, which could interfere with bioassays for IL-13,the supernatant was treated with biotinylated antibodies to GM-CSF andIL-4 (R&D Systems, Inc), then incubated with streptavidin-coatedmagnetic beads (Dynal). The final concentration of IL-13 was determinedby ELISA (Biosource), and for total protein by Bradford assay (Bio-Rad).The typical preparation contains <0.0005% IL-13 by weight.

Selection of Hybridoma Clones

Using established methods, hybridomas were generated from spleens ofmice selected as above, fused to the P3X63_AG8.653 myeloma cell line(ATCC). Cells were plated at limiting dilution and clones were selectedaccording to the screening criteria described above. Data was collectedfor the selection of clones based on ability to compete for NHP-IL-13binding to sIL13Rα1-Fc by ELISA. Clones were further tested for abilityto neutralize the bioactivity of NHP-IL-13. Supernatants of thehybridomas were tested for competition of STAT-6 phosphorylation inducedby NHP-IL-13 in the HT-29 human epithelial cell line.

Example 2 MJ 2-7 Antibody

Total RNA was prepared from MJ 2-7 hybridoma cells using the QIAGENRNEASY3 Mini Kit (Qiagen). RNA was reverse transcribed to cDNA using theSMART3 PCR Synthesis Kit (BD Biosciences Clontech). The variable regionof MJ 2-7 heavy chain was extrapolated by PCR using SMART3oligonucleotide as a forward primer and mIgG1 primer annealing to DNAencoding the N-terminal part of CH1 domain of mouse IgG1 constant regionas a reverse primer. The DNA fragment encoding MJ 2-7 light chainvariable region was generated using SMART3 and mouse kappa specificprimers. The PCR reaction was performed using DEEP VENT3 DNA polymerase(New England Biolabs) and 25 nM of dNTPs for 24 cycles (94° C. for 1minute, 60° C. for 1 minute, 72° C. for 1 minute). The PCR products weresubcloned into the pED6 vector, and the sequence of the inserts wasidentified by DNA sequencing. N-terminal protein sequencing of thepurified mouse MJ 2-7 antibody was used to confirm that the translatedsequences corresponded to the observed protein sequence.

Exemplary nucleotide and amino acid sequences of mouse monoclonalantibody MJ 2-7 which interacts with NHP IL-13 and which hascharacteristics which suggest that it may interact with human IL-13 areas follows:

An exemplary nucleotide sequence encoding the heavy chain variabledomain includes:

(SEQ ID NO: 129) GAG GTTCAGCTGC AGCAGTCTGG GGCAGAGCTT GTGAAGCCAGGGGCCTCAGT CAAGTTGTCC TGCACAGGTT CTGGCTTCAACATTAAAGAC ACCTATATAC ACTGGGTGAA GCAGAGGCCTGAACAGGGCC TGGAGTGGAT TGGAAGGATT GATCCTGCGAATGATAATAT TAAATATGAC CCGAAGTTCC AGGGCAAGGCCACTATAACA GCAGACACAT CCTCCAACAC AGCCTACCTACAGCTCAACA GCCTGACATC TGAGGACACT GCCGTCTATTACTGTGCTAG ATCTGAGGAA AATTGGTACG ACTTTTTTGACTACTGGGGC CAAGGCACCA CTCTCACAGT CTCCTCA

An exemplary amino acid sequence for the heavy chain variable domainincludes:

(SEQ ID NO: 130) EVQLQQSGAELVKPGASVKLSCTGS GFNIKDTYIH WVKQRPEQGLEWI GRIDPANDNIKYDPKFQG KATITADTSSNTAYLQLNSLTSEDTAVYYC AR SEENWYDFFDYWGQGTTLTVSS

CDRs are underlined. The variable domain optionally is preceded by aleader sequence. e.g., MKCSWVIFFLMAVVTGVNS (SEQ ID NO:131). An exemplarynucleotide sequence encoding the light chain variable domain includes:

(SEQ ID NO: 132) GAT GTTTTGATGA CCCAAACTCC ACTCTCCCTG CCTGTCAGTCTTGGAGATCA AGCCTCCATC TCTTGCAGGT CTAGTCAGAGCATTGTACAT AGTAATGGAA ACACCTATTT AGAATGGTACCTGCAGAAAC CAGGCCAGTC TCCAAAGCTC CTGATCTACAAAGTTTCCAA CCGATTTTCT GGGGTCCCAG ACAGGTTCAGTGGCAGTGGA TCAGGGACAG ATTTCACACT CAAGATTAGCAGAGTGGAGG CTGAGGATCT GGGAGTTTAT TACTGCTTTCAAGGTTCACA TATTCCGTAC ACGTTCGGAG GGGGGACCAA GCTGGAAATA AAA

An exemplary amino acid sequence for the light chain variable domainincludes:

(SEQ ID NO: 133) DVLMTQTPLSLPVSLGDQASISC RSSQSIVHSNGNTYLE WYLQKPGQSPKLLIY KVSNRFSG VPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSH IPYT FGGGTKLEIK

CDRs are underlined. The amino acid sequence optionally is preceded by aleader sequence, e.g., MKLPVRLLVLMFWIPASSS (SEQ ID NO:134). The term “MJ2-7” is used interchangeably with the term “mAb7.1.1,” herein.

Example 3 C65 Antibody

Exemplary nucleotide and amino acid sequences of mouse monoclonalantibody C65, which interacts with NHP IL-13 and which hascharacteristics that suggest that it may interact with human IL-13 areas follows:

An exemplary nucleic acid sequence for the heavy chain variable domainincludes:

(SEQ ID NO: 135)  1 ATGGCTGTCC TGGCATTACT CTTCTGCCTG GTAACATTCC CAAGCTGTAT 51 CCTTTCCCAG GTGCAGCTGA AGGAGTCAGG ACCTGGCCTG GTGGCGCCCT101 CACAGAGCCT GTCCATCACA TGCACCGTCT CAGGGTTCTC ATTAACCGGC151 TATGGTGTAA ACTGGGTTCG CCAGCCTCCA GGAAAGGGTC TGGAGTGGCT201 GGGAATAATT TGGGGTGATG GAAGCACAGA CTATAATTCA GCTCTCAAAT251 CCAGACTGAT CATCAACAAG GACAACTCCA AGAGCCAAGT TTTCTTAAAA301 ATGAACAGTC TGCAAACTGA TGACACAGCC AGGTACTTCT GTGCCAGAGA351 TAAGACTTTT TACTACGATG GTTTCTACAG GGGCAGGATG GACTACTGGG401 GTCAAGGAAC CTCAGTCACC GTCTCCTCA

An exemplary amino acid sequence for the heavy chain variable domainincludes:

(SEQ ID NO: 136) QVQLKESGPGL VAPSQSLSIT CTVS GFSLTG   YGVN WVRQPPGKGLEWLG II   WGDGSTDYNS   AL KSRLIINK DNSKSQVFLK MNSLQTDDTA RYFCAR DKTF  YYDGFYRGRM   DY WGQGTSVT VSSCDRs are underlined. The amino acid sequence optionally is preceded by aleader sequence, e.g., MAVLALLFCL VTFPSCILS (SEQ ID NO:137).

An exemplary nucleotide sequence encoding the light chain variabledomain includes:

(SEQ ID NO: 138)  1 ATGAACACGA GGGCCCCTGC TGAGTTCCTT GGGTTCCTGT TGCTCTGGTT 51 TTTAGGTGCC AGATGTGATG TCCAGATGAT TCAGTCTCCA TCCTCCCTGT101 CTGCATCTTT GGGAGACATT GTCACCATGA CTTGCCAGGC AAGTCAGGGC151 ACTAGCATTA ATTTAAACTG GTTTCAGCAA AAACCAGGGA AAGCTCCTAA201 GCTCCTGATC TTTGGTGCAA GCAACTTGGA AGATGGGGTC CCATCAAGGT251 TCAGTGGCAG TAGATATGGG ACAAATTTCA CTCTCACCAT CAGCAGCCTG301 GAGGATGAAG ATATGGCAAC TTATTTCTGT CTACAGCATA GTTATCTCCC351 GTGGACGTTC GGTGGCGGCA CCAAACTGGA AATCAAA

An exemplary amino acid sequence for the light chain variable domainincludes:

(SEQ ID NO: 139) DVQMIQSP SSLSASLGDI VTMTC QASCQG TSINLN WFQQKPGKAPKLLI F GASNLED GV PSRFSGSRYG TNFTLTISSL EDEDMATYFC  LQHSYLPWTF GGGTKLEIKCDRs are underlined. The amino acid sequence optionally is preceded by aleader sequence, e.g., MNTRAPAEFLGFLLLWFLGARC (SEQ ID NO:140).

Example 4 Cynomolgus Monkey Model

The efficacy of an antibody to neutralize one or more IL-13-associatedactivities in vivo can be tested using a model of antigen-induced airwayinflammation in cynomolgus monkeys naturally allergic to Ascaris suum.In this model, challenge of an allergic monkey with Ascaris suum antigenresults in an influx of inflammatory cells, especially eosinophils, intothe airways. To test the ability of an antibody to prevent this influxof cells, the antibody can be administered 24 hours prior to challengewith Ascaris suum antigen. On the day of challenge, a baselinebronchoalveolar lavage (BAL) sample can be taken from the left lung. Theantigen can then be instilled intratracheally into the right lung.Twenty-four hours later, the right lung is lavaged, and the BAL fluidfrom animals treated intravenously with 10 mg/kg recombinant antibodyexpressed from CHO cells are compared to BAL fluid from untreatedanimals. If the antibody reduces airway inflammation, an increase inpercent BAL eosinophils may be observed among the untreated group, butnot for the antibody-treated group. These assays can be used to confirmthat the antibody effectively prevents airway eosinophilia in allergicanimals challenged with an allergen.

Example 5 Fc Sequences

The Ser at position #1 of SEQ ID NO:128 represents amino acid residue#119 in a first exemplary full length antibody numbering scheme in whichthe Ser is preceded by residue #118 of a heavy chain variable domain. Inthe first exemplary full length antibody numbering scheme, mutated aminoacids are at numbered 234 and 237, and correspond to positions 116 and119 of SEQ ID NO:128. Thus, the following sequence represents an Fcdomain with two mutations: L234A and G237A, according to the firstexemplary full length antibody numbering scheme.

Mus musculus (SEQ ID NO:128)

The following is another exemplary human Fc domain sequence:

(SEQ ID NO: 141) STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Other exemplary alterations that can be used to decrease effectorfunction include L234A;L235A), (L235A;G237A), and N297A.

Example 6 IL-13 and IgE in Mice

IL-13 is involved in the production of IgE, an important mediator ofatopic disease. Mice deficient in IL-13 had partial reductions in serumIgE and mast cell IgE responses, whereas mice lacking the natural IL-13binding agent, IL-13Rα2−/−, had enhanced levels of IgE and IgE effectorfunction.

BALB/c female mice were obtained from Jackson Laboratories (Bar Harbor,Me.). IL-13RI2−/− mice are described, e.g., in Wood et al. (2003) J.Exp. Med. 197:703-9. Mice deficient in IL-13 are described, e.g., inMcKenzie et al. (1998) Immunity 9:423-32. All mutant strains were on theBALB/c background.

Serum IgE levels were measured by ELISA. ELISA plates (MaxiSorp; Nunc,Rochester, N.Y.) were coated overnight at 4° C. with rat anti-mouse IgE(BD Biosciences, San Diego, Calif.). Plates were blocked for 1 hour atroom temperature with 0.5% gelatin in PBS, washed in PBS containing0.05% TWEEN®-20 (PBS-Tween), and incubated for six hours at roomtemperature with purified mouse IgE (BD Biosciences) as standards orwith serum dilutions. Binding was detected with biotinylated anti-mouseIgE (BD Biosciences) using mouse IgG (Sigma-Aldrich, St. Louis, Mo.) asa blocker. Binding was detected with peroxidase-linked streptavidin(Southern Biotechnology Associates, Inc., Birmingham, Ala.) and SUREBLUE3 substrate (KPL Inc., Gaithersburg, Md.).

In order to investigate the requirement for IL-13 to support resting IgElevels in naive mice, serum was examined in the absence of specificimmunization from wild-type mice and from mice genetically deficient inIL-13 and IL-13Rα2. Mice deficient in IL-13 had virtually undetectablelevels of serum IgE. In contrast, mice lacking the inhibitory receptorIL-13Rα2 displayed elevated levels of serum IgE. These resultsdemonstrate that blocking IL-13 can be useful for treating or preventingatopic disorders.

Example 7 IL-13 and Atopic Disorders

The ability of MJ2-7 to inhibit the bioactivity of native human IL-13(at 1 ng/ml) was evaluated in an assay for STATE phosphorylation. MJ2-7inhibited the activity of native human IL-13 with an IC50 of about 0.293nM in this assay. An antibody with the murine heavy chain of MJ2-7 and ahumanized light chain inhibited the activity of native human IL-13 withan IC50 of about 0.554 nM in this assay.

The ability of MJ2-7 to inhibit non-human primate IL-13 (at 1 ng/ml) wasevaluated in an assay for CD23 expression. The MJ2-7 inhibited theactivity of non-human primate IL-13 with an IC50 of about 0.242 nM inthis assay. An antibody with the murine heavy chain of MJ2-7 and ahumanized light chain inhibited the activity of non-human primate IL-13with an IC50 of about 0.308 nM in this assay.

Example 8 Nucleotide and Amino Acid Sequences of Mouse MJ 2-7 Antibody

The nucleotide sequence encoding the heavy chain variable region (withan optional leader) is as follows:

(SEQ ID NO: 142)  1 ATGAAATGCA GCTGGGTTAT CTTCTTCCTG ATGGCAGTGG TTACAGGGGT 51 CAATTCAGAG GTTCAGCTGC AGCAGTCTGG GGCAGAGCTT GTGAAGCCAG101 GGGCCTCAGT CAAGTTGTCC TGCACAGGTT CTGGCTTCAA CATTAAAGAC151 ACCTATATAC ACTGGGTGAA GCAGAGGCCT GAACAGGGCC TGGAGTGGAT201 TGGAAGGATT GATCCTGCGA ATGATAATAT TAAATATGAC CCGAAGTTCC251 AGGGCAAGGC CACTATAACA GCAGACACAT CCTCCAACAC AGCCTACCTA301 CAGCTCAACA GCCTGACATC TGAGGACACT GCCGTCTATT ACTGTGCTAG351 ATCTGAGGAA AATTGGTACG ACTTTTTTGA CTACTGGGGC CAAGGCACCA401 CTCTCACAGT CTCCTCA

The amino acid sequence of the heavy chain variable region with anoptional leader (underscored) is as follows:

(SEQ ID NO: 143)  1 MKCSWVIFFL MAVVTGVNSE VQLQQSGAEL VKPGASVKLS CTGSGFNIKD 51 TYIHWVKQRP EQGLEWIGRI DPANDNIKYD PKFQGKATIT ADTSSNTAYL101 QLNSLTSEDT AVYYCARSEE NWYDFFDYWG QGTTLTVSS

The nucleotide sequence encoding the light chain variable region is asfollows:

(SEQ ID NO: 144)  1 ATGAAGTTGC CTGTTAGGCT GTTGGTGCTG ATGTTCTGGA TTCCTGCTTC 51 CAGCAGTGAT GTTTTGATGA CCCAAACTCC ACTCTCCCTG CCTGTCAGTC101 TTGGAGATCA AGCCTCCATC TCTTGCAGGT CTAGTCAGAG CATTGTACAT151 AGTAATGGAA ACACCTATTT AGAATGGTAC CTGCAGAAAC CAGGCCAGTC201 TCCAAAGCTC CTGATCTACA AAGTTTCCAA CCGATTTTCT GGGGTCCCAG251 ACAGGTTCAG TGGCAGTGGA TCAGGGACAG ATTTCACACT CAAGATTAGC301 AGAGTGGAGG CTGAGGATCT GGGAGTTTAT TACTGCTTTC AAGGTTCACA351 TATTCCGTAC ACGTTCGGAG GGGGGACCAA GCTGGAAATA AAA 

The amino acid sequence of the light chain variable region with anoptional leader (underscored) is as follows:

(SEQ ID NO: 145)  1 MKLPVRLLVL MFWIPASSSD VLMTQTPLSL PVSLGDQASI SCRSSQSIVH 51 SNGNTYLEWY LQKPGQSPKL LIYKVSNRFS GVPDRFSGSG SGTDFTLKIS101 RVEAEDLGVY YCFQGSHIPY TFGGGTKLEI K

Example 9 Nucleotide and Amino Acid Sequences of Exemplary FirstHumanized Variants of the MJ 2-7 Antibody

Humanized antibody Version 1 (V1) is based on the closest human germlineclones. The nucleotide sequence of hMJ 2-7 V1 heavy chain variableregion (hMJ 2-7 VH V1) (with a sequence encoding an optional leadersequence) is as follows:

(SEQ ID NO: 146)  1 ATGGATTGGA CCTGGCGCAT CCTGTTCCTG GTGGCCGCTG CCACCGGCGC 51 TCACTCTCAG GTGCAGCTGG TGCAGTCTGG CGCCGAGGTG AAGAAGCCTG101 GCGCTTCCGT GAAGGTGTCC TGTAAGGCCT CCGGCTTCAA CATCAAGGAC151 ACCTACATCC ACTGGGTGCG GCAGGCTCCC GGCCAGCGGC TGGAGTGGAT201 GGGCCGGATC GATCCTGCCA ACGACAACAT CAAGTACGAC CCCAAGTTTC251 AGGGCCGCGT GACCATCACC CGCGATACCT CCGCTTCTAC CGCCTACATG301 GAGCTGTCTA GCCTGCGGAG CGAGGATACC GCCGTGTACT ACTGCGCCCG351 CTCCGAGGAG AACTGGTACG ACTTCTTCGA CTACTGGGGC CAGGGCACCC401 TGGTGACCGT GTCCTCT

The amino acid sequence of the heavy chain variable region (hMJ 2-7 V1)is based on a CDR grafted to DP-25, VH-I, 1-03. The amino acid sequencewith an optional leader (first underscored region; CDRs based on AbMdefinition shown in subsequent underscored regions) is as follows:

(SEQ ID NO: 147)  1 MDWTWRILFL VAAATGAHS-Q VQLVQSGAEV KKPGASVKVS CKASGFNIKD 51 TYIHWVRQAP GQRLEWMGRI DPANDNIKYD PKFQGRVTIT RDTSASTAYM101 ELSSLRSEDT AVYYCARSEE NWYDFFDYWG QGTLVTVSSG ESCR

The nucleotide sequence of the hMJ 2-7 V1 light chain variable region(hMJ 2-7 VL V1) (with a sequence encoding an optional leader sequence)is as follows:

(SEQ ID NO: 148)  1 ATGCGGCTGC CCGCTCAGCT GCTGGGCCTG CTGATGCTGT GGGTGCCCGG 51 CTCTTCCGGC GACGTGGTGA TGACCCAGTC CCCTCTGTCT CTGCCCGTGA101 CCCTGGGCCA GCCCGCTTCT ATCTCTTGCC GGTCCTCCCA GTCCATCGTG151 CACTCCAACG GCAACACCTA CCTGGAGTGG TTTCAGCAGA GACCCGGCCA201 GTCTCCTCGG CGGCTGATCT ACAAGGTGTC CAACCGCTTT TCCGGCGTGC251 CCGATCGGTT CTCCGGCAGC GGCTCCGGCA CCGATTTCAC CCTGAAGATC301 AGCCGCGTGG AGGCCGAGGA TGTGGGCGTG TACTACTGCT TCCAGGGCTC351 CCACATCCCT TACACCTTTG GCGGCGGAAC CAAGGTGGAG ATCAAG

This version is based on a CDR graft to DPK18, V kappaII. The amino acidsequence of hMJ 2-7 V1 light chain variable region (hMJ 2-7 VL V1) (withoptional leader as first underscored region; CDRs based on AbMdefinition in subsequent underscored regions) is as follows:

(SEQ ID NO: 149)   1 MRLPAQLLGL LMLWVPGSSG-DVVMTQSPLS LPVTLGQPAS ISCRSSQSIV  51  HSNGNTYLE W FQQRPGQSPR RLIY KVSNRF S GVPDRFSGS GSGTDFTLKI101 SRVEAEDVGV YYC FQGSHIP   YT FGGGTKVE IK

Example 10 Nucleotide and Amino Acid Sequences of Exemplary SecondHumanized Variants of the MJ 2-7 Antibody

The following heavy chain variable region is based on a CDR graft toDP-54, VH-3, 3-07. The nucleotide sequence of hMJ 2-7 Version 2 (V2)heavy chain variable region (hMJ 2-7 VH V2) (with a sequence encoding anoptional leader sequence) is as follows:

(SEQ ID NO: 150)  1 ATGGAGCTGG GCCTGTCTTG GGTGTTCCTG GTGGCTATCC TGGAGGGCGT 51 GCAGTGCGAG GTGCAGCTGG TGGAGTCTGG CGGCGGACTG GTGCAGCCTG101 GCGGCTCTCT GCGGCTGTCT TGCGCCGCTT CCGGCTTCAA CATCAAGGAC151 ACCTACATCC ACTGGGTGCG GCAGGCTCCC GGCAAGGGCC TGGAGTGGGT201 GGCCCGGATC GATCCTGCCA ACGACAACAT CAAGTACGAC CCCAAGTTCC251 AGGGCCGGTT CACCATCTCT CGCGACAACG CCAAGAACTC CCTGTACCTC301 CAGATGAACT CTCTGCGCGC CGAGGATACC GCCGTGTACT ACTGCGCCCG351 GAGCGAGGAG AACTGGTACG ACTTCTTCGA CTACTGGGGC CAGGGCACCC401 TGGTGACCGT GTCCTCT

The amino acid sequence of hMJ 2-7 V2 heavy chain variable region (hMJ2-7 VH V2) with an optional leader (first underscored region; CDRs basedon AbM definition shown in subsequent underscored regions) is asfollows:

(SEQ ID NO: 151)   1 MELGLSWVFL VAILEGVQC- E VQLVESGGGL VQPGGSLRLS CAASGFNIKD  51 TYIH WVRQAP GKGLEWVA RI   DPANDNIKYD PKFQG RFTIS RDNAKNSLYL101 QMNSLRAEDT AVYYCAR SEE   NWYDFFDY WG QGTLVTVSS

The hMJ 2-7 V2 light chain variable region was based on a CDR graft toDPK9, V kappaI, 02. The nucleotide sequence of hMJ 2-7 V2 light chainvariable region (hMJ 2-7 VL V2) (with a sequence encoding an optionalleader sequence) is as follows:

(SEQ ID NO: 152)   1ATGGATATGC GCGTGCCCGC TCAGCTGCTG GGCCTGCTGC TGCTGTGGCT  51GCGCGGAGCC CGCTGCGATA TCCAGATGAC CCAGTCCCCT TCTTCTCTGT 101CCGCCTCTGT GGGCGATCGC GTGACCATCA CCTGTCGGTC CTCCCAGTCC 151ATCGTGCACT CCAACGGCAA CACCTACCTG GAGTGGTATC AGCAGAAGCC 201CGGCAAGGCC CCTAAGCTGC TGATCTACAA GGTGTCCAAC CGCTTTTCCG 251GCGTGCCTTC TCGGTTCTCC GGCTCCGGCT CCGGCACCGA TTTCACCCTG 301ACCATCTCCT CCCTCCAGCC CGAGGATTTC GCCACCTACT ACTGCTTCCA 351GGGCTCCCAC ATCCCTTACA CCTTTGGCGG CGGAACCAAG GTGGAGATCA 401 AGCGT

The amino acid sequence of the light chain variable region of hMJ 2-7 V2light chain variable region (hMJ 2-7 VL V2) (with optional leaderpeptide underscored and CDRs based on AbM definition shown in subsequentunderscored regions) is as follows:

(SEQ ID NO: 153)   1 MDMRVPAQLL GLLLLWLRGA RC -DIQMTQSP SSLSASVGDR VTITCRSSQS  51 IVHSNGNTYL E WYQQKPGKA PKLLIY KVSN   RFS GVPSRFS GSGSGTDFTL101 TISSLQPEDF ATYYC FQGSH   IPYT FGGGTK VEIKR

Additional humanized versions of MJ 2-7 V2 heavy chain variable regionwere made. These versions included backmutations that have murine aminoacids at selected framework positions.

The nucleotide sequence encoding the heavy chain variable region“Version 2.1” or V2.1 with the back mutations V48I,A29G is as follows:

(SEQ ID NO: 154)   1GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC  51TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA 101TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GATCGGCCGG 151ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG 201GTTCACCATC TCTCGCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA 251ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG 301GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC 351 CGTGTCCTCT

The amino acid sequence of the heavy chain variable region of V2.1 (CDRsbased on AbM definition shown in subsequent underscored regions) is asfollows:

(SEQ ID NO: 155)   1 EVQLVESGGG LVQPGGSLRL SCAAS GFNIK DTYIHWVRQA PGKGLEWIG R  51 IDPANDNIKY DPKFQGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR SE 101 ENWYDFFDY W GQGTLVTVSS

The nucleotide sequence encoding the heavy chain variable region V2.2with the back mutations (R67K;F68A) is as follows:

(SEQ ID NO: 156)   1GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC  51TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA 101TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGCCCGG 151ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCAA 201GGCCACCATC TCTCGCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA 251ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG 301GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC 351 CGTGTCCTCT

The amino acid sequence of the heavy chain variable region of V2.2 (CDRsbased on AbM definition shown in subsequent underscored regions) is asfollows:

(SEQ ID NO: 157)   1 EVQLVESGGG LVQPGGSLRL SCAAS GFNIK DTYIHWVRQA PGKGLEWVA R  51 IDPANDNIKY DPKFQGKATI SRDNAKNSLY LQMNSLRAED TAVYYCAR SE 102 ENWYDFFDY W GQGTLVTVSS

The nucleotide sequence encoding the heavy chain variable region V2.3with the back mutations (R72A):

(SEQ ID NO: 158)   1GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC  51TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA 101TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGCCCGG 151ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG 201GTTCACCATC TCTGCCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA 251ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG 301GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC 351 CGTGTCCTCT

The amino acid sequence of the heavy chain variable region of V2.3 (CDRsbased on AbM definition shown in subsequent underscored regions) is asfollows:

(SEQ ID NO: 159)   1 EVQLVESGGG LVQPGGSLRL SCAAS GFNIK DTYIHWVRQA PGKGLEWVA R  51 IDPANDNIKY DPKFQGRFTI SADNAKNSLY LQMNSLRAED TAVYYCAR SE 103 ENWYDFFDY W GQGTLVTVSS

The nucleotide sequence encoding the heavy chain variable region V2.4with the back mutations (A49G) is as follows:

(SEQ ID NO: 160)   1GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC  51TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA 101TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGGCCGG 151ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG 201GTTCACCATC TCTCGCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA 251ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG 301GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC 351 CGTGTCCTCT

The amino acid sequence of the heavy chain variable region of V2.4 (CDRsbased on AbM definition shown in subsequent underscored regions) is asfollows:

(SEQ ID NO: 161)   1 EVQLVESGGG LVQPGGSLRL SCAAS GFNIK DTYIHWVRQA PGKGLEWVG R  51 IDPANDNIKY DPKFQGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR SE 104 ENWYDFFDY W GQGTLVTVSS

The nucleotide sequence encoding the heavy chain variable region V2.5with the back mutations (R67K;F68A;R72A) is as follows:

(SEQ ID NO: 162)   1GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC  51TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA 101TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGCCCGG 151ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCAA 201GGCCACCATC TCTGCCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA 251ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG 301GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC 352 CGTGTCCTCT

The amino acid sequence of the heavy chain variable region of V2.5 (CDRsbased on AbM definition shown in subsequent underscored regions) is asfollows:

(SEQ ID NO: 163)   1 EVQLVESGGG LVQPGGSLRL SCAAS GFNIK DTYIHWVRQA PGKGLEWVA R  51 IDPANDNIKY DPKFQGKATI SADNAKNSLY LQMNSLRAED TAVYYCAR SE 105 ENWYDFFDY W GQGTLVTVSS

The nucleotide sequence encoding the heavy chain variable region V2.6with the back mutations (V48I;A49G;R72A) is as follows:

(SEQ ID NO: 164)   1GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC  51TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA 101TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GATCGGCCGG 151ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG 201GTTCACCATC TCTGCCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA 251ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG 301GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC 351 CGTGTCCTCT

The amino acid sequence of the heavy chain variable region of V2.6 (CDRsbased on AbM definition shown in subsequent underscored regions) is asfollows:

(SEQ ID NO: 165)   1 EVQLVESGGG LVQPGGSLRL SCAAS GFNIK DTYIHWVRQA PGKGLEWIG R  51 IDPANDNIKY DPKFQGRFTI SADNAKNSLY LQMNSLRAED TAVYYCAR SE 106 ENWYDFFDY W GQGTLVTVSS

The nucleotide sequence encoding the heavy chain variable region V2.7with the back mutations (A49G;R72A) is as follows:

(SEQ ID NO: 166) 1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGCCTGGCGGCTC 51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA 101TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGGCCGG 151ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG 201GTTCACCATC TCTGCCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA 251ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG 301GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC 351 CGTGTCCTCT

The amino acid sequence of the heavy chain variable region of V2.7 (CDRsbased on AbM definition shown in subsequent underscored regions) is asfollows:

(SEQ ID NO: 167) 1 EVQLVESGGG LVQPGGSLRL SCAAS GFNIK DTYIH WVRQAPGKGLEWVG R 51 IDPANDNIKY DPKFQG RFTI SADNAKNSLY LQMNSLRAED TAVYYCAR SE107 ENWYDFFDY W GQGTLVTVSS

The nucleotide sequence encoding the heavy chain variable region V2.8with the back mutations (L79A) is as follows:

(SEQ ID NO: 168) 1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGCCTGGCGGCTC 51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA 101TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGCCCGG 151ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG 201GTTCACCATC TCTCGCGACA ACGCCAAGAA CTCCGCCTAC CTCCAGATGA 251ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG 301GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC 351 CGTGTCCTCT

The amino acid sequence of the heavy chain variable region of V2.8

(CDRs based on AbM definition shown in subsequent underscored regions)is as follows:

(SEQ ID NO: 169) 1 EVQLVESGGG LVQPGGSLRL SCAAS GFNIK DTYIH WVRQAPGKGLEWVA R 51 IDPANDNIKY DPKFQG RFTI SRDNAKNSAY LQMNSLRAED TAVYYCAR SE108 ENWYDFFDY W GQGTLVTVSS

The nucleotide sequence encoding the heavy chain variable region V2.10with the back mutations (A49G;R72A;L79A) is as follows:

(SEQ ID NO: 170) 1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGCCTGGCGGCTC 51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA 101TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGGCCGG 151ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG 201GTTCACCATC TCTGCCGACA ACGCCAAGAA CTCCGCCTAC CTCCAGATGA 251ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG 301GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC 351 CGTGTCCTCT

The amino acid sequence of the heavy chain variable region of V2.10(CDRs based on AbM definition shown in subsequent underscored regions)is as follows:

(SEQ ID NO: 171) 1 EVQLVESGGG LVQPGGSLRL SCAAS GFNIK DTYIH WVRQAPGKGLEWVG R 51 IDPANDNIKY DPKFQG RFTI SADNAKNSAY LQMNSLRAED TAVYYCAR SE109 ENWYDFFDY W GQGTLVTVSS

The nucleotide sequence encoding the heavy chain variable region V2.11with the back mutations (V48I;A49G;R72A;L79A) is as follows:

(SEQ ID NO: 172) 1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGCCTGGCGGCTC 51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA 101TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GATCGGCCGG 151ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG 201GTTCACCATC TCTGCCGACA ACGCCAAGAA CTCCGCCTAC CTCCAGATGA 251ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG 301GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC 351 CGTGTCCTCT

The amino acid sequence of the heavy chain variable region of V2.11(CDRs based on AbM definition shown in subsequent underscored regions)is as follows:

(SEQ ID NO: 173) 1 EVQLVESGGG LVQPGGSLRL SCAAS GFNIK DTYIH WVRQAPGKGLEWIG R 51 IDPANDNIKY DPKFQG RFTI SADNAKNSAY LQMNSLRAED TAVYYCAR SE110 ENWYDFFDY W GQGTLVTVSS

The nucleotide sequence encoding the heavy chain variable region V2.16with the back mutations (V48I;A49G;R72A) is as follows:

(SEQ ID NO: 174) 1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGCCTGGCGGCTC 51 TCTGCGGCTG TCTTGCACCG GCTCCGGCTT CAACATCAAG GACACCTACA 101TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GATCGGCCGG 151ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG 201GTTCACCATC TCTGCCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA 251ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG 301GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC 351 CGTGTCCTCT

The amino acid sequence of the heavy chain variable region of V2.16(CDRs based on AbM definition shown in subsequent underscored regions)is as follows:

(SEQ ID NO: 175) 1 EVQLVESGGG LVQPGGSLRL SCTGS GFNIK DTYIH WVRQAPGKGLEWIG R 51 IDPANDNIKY DPKFQG RFTI SADNAKNSLY LQMNSLRAED TAVYYCAR SE111 ENWYDFFDY W GQGTLVTVSS

The following is the amino acid sequence of a humanized MH 2-7 V2.11IgG1 with a mutated CH2 domain:

(SEQ ID NO: 176) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWIGRIDPANDNIKYDPKFQGRFTISADNAKNSAYLQMNSLRAEDTAVYYCARSEENWYDFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPE A LG APSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The variable domain is at amino acids 1-120; CH1 at 121-218; hinge at219-233; CH2 at 234-343; and CH3 at 344-450. The light chain includesthe following sequence with variable domain at 1-133.

(SEQ ID NO: 177) DIQMTQSPSSLSASVGDRVTITCRSSQSIVHSNGNTYLEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSHIPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 11 Functional Assays of Exemplary Variants of MJ2-7

We evaluated the ability of the MJ2-7 antibody and humanized variants toinhibit human IL-13 in assays for IL-13 activity.

STAT6 Phosphorylation Assay.

HT-29 human colonic epithelial cells (ATCC) were grown as an adherentmonolayer in McCoy's 5A medium containing 10% FBS, Pen-Strep, glutamine,and sodium bicarbonate. For assay, the cells were dislodged from theflask using trypsin, washed into fresh medium, and distributed into12×75 mm polystyrene tubes. Recombinant human IL-13 (R&D Systems, Inc.)was added at concentrations ranging from 100-0.01 ng/ml. For assaystesting the ability of antibody to inhibit the IL-13 response, 1 ng/mlrecombinant human IL-13 was added along with dilutions of antibodyranging from 500-0.4 ng/ml. Cells were incubated in a 37° C. water bathfor 30-60 minutes, then washed into ice-cold PBS containing 1% BSA.Cells were fixed by incubating in 1% paraformaldehyde in PBS for 15minutes at 37° C., then washed into PBS containing 1% BSA. Topermeabilize the nucleus, cells were incubated overnight at −20° C. inabsolute methanol. They were washed into PBS containing 1% BSA, thenstained with ALEXA3 Fluor 488-labeled antibody to STATE (BDBiosciences). Fluorescence was analyzed with a FACSCAN3 and CELLQUEST3software (BD Biosciences).

CD23 Induction on Human Monocytes

Mononuclear cells were isolated from human peripheral blood by layeringover HISTOPAQUE® (Sigma). Cells were washed into RPMI containing 10%heat-inactivated FCS, 50 U/ml penicillin, 50 mg/ml streptomycin, 2 mML-glutamine, and plated in a 48-well tissue culture plate(Costar/Corning). Recombinant human IL-13 (R&D Systems, Inc.) was addedat dilutions ranging from 100-0.01 ng/ml. For assays testing the abilityof antibody to inhibit the IL-13 response, 1 ng/ml recombinant humanIL-13 was added along with dilutions of antibody ranging from 500-0.4ng/ml. Cells were incubated overnight at 37° C. in a 5% CO₂ incubator.The next day, cells were harvested from wells using non-enzymatic CellDissociation Solution (Sigma), then washed into ice-cold PBS containing1% BSA. Cells were incubated with phycoerythrin (PE)-labeled antibody tohuman CD23 (BD Biosciences, San Diego, Calif.), and Cy-Chrome-labeledantibody to human CD11b (BD Biosciences). Monocytes were gated based onhigh forward and side light scatter, and expression of CD11b. CD23expression on monocytes was determined by flow cytometry using aFACSCAN3 (BD Biosciences), and the percentage of CD23⁺ cells wasanalyzed with CELLQUEST3 software (BD Biosciences).

TF-1 Cell Proliferation

TF-1 cells are a factor-dependent human hemopoietic cell line requiringinterleukin 3 (IL-3) or granulocyte/macrophage colony-stimulating factor(GM-CSF) for their long-term growth. TF-1 cells also respond to avariety of other cytokines, including interleukin 13 (IL-13). TF-1 cells(ATCC) were maintained in RPMI medium containing 10% heat-inactivatedFCS, 50 U/ml penicillin, 50 mg/ml streptomycin, 2 mM L-glutamine, and 5ng/ml recombinant human GM-CSF (R&D Systems). Prior to assay, cells werestarved of GM-CSF overnight. For assay, TF-1 cells were plated induplicate at 5000 cells/well in 96-well flat-bottom microtiter plates(Costar/Corning), and challenged with human IL-13 (R&D Systems), rangingfrom 100-0.01 ng/ml. After 72 hours in a 37° C. incubator with 5% CO₂,the cells were pulsed with 1 TCi/well ³H-thymidine (Perkin Elmer/NewEngland Nuclear). They were incubated an additional 4.5 hours, thencells were harvested onto filter mats using a TOMTEK3 harvester.³H-thymidine incorporation was assessed by liquid scintillationcounting.

Tenascin Production Assay

BEAS-2B human bronchial epithelial cells (ATCC) were maintained BEGMmedia with supplements (Clonetics). Cells were plated at 20,000 per wellin a 96-well flat-bottom culture plate overnight. Fresh media is addedcontaining IL-13 in the presence or absence of the indicated antibody.After overnight incubation, the supernatants are harvested, and assayedfor the presence of the extracellular matrix component, tenascin C, byELISA. ELISA plates are coated overnight with 1 ug/ml of murinemonoclonal antibody to human tenascin (IgG1, k; Chemicon International)in PBS. Plates are washed with PBS containing 0.05% TWEEN®-20(PBS-Tween), and blocked with PBS containing 1% BSA. Fresh blockingsolution was added every 6 minutes for a total of three changes. Plateswere washed 3× with PBS-Tween. Cell supernatants or human tenascinstandard (Chemicon International) were added and incubated for 60minutes at 37° C. Plates were washed 3× with PBS-Tween. Tenascin wasdetected with murine monoclonal antibody to tenascin (IgG2a, k; Biohit).Binding was detected with HRP-labeled antibody to mouse IgG2a, followedby TMB substrate. The reaction was stopped with 0.01 N sulfuric acid.Absorbance was read at 450 nm.

The HT 29 human epithelial cell line can be used to assay STAT6phosphorylation. HT 29 cells are incubated with 1 ng/ml native humanIL-13 crude preparation in the presence of increasing concentrations ofthe test antibody for 30 minutes at 37° C. Western blot analysis of celllysates with an antibody to phosphorylated STAT6 can be used to detectdose-dependent IL 13-mediated phosphorylation of STAT6. Similarly, flowcytometric analysis can detect phosphorylated STAT6 in HT 29 cells thatwere treated with a saturating concentration of IL-13 for 30 minutes at37° C., fixed, permeabilized, and stained with an ALEXA™ Fluor488-labeled mAb to phospho-STAT6. An exemplary set of results is setforth in the Table 1. The inhibitory activity of V2.11 was comparable tothat of sIL-13Rα2-Fc.

TABLE 1 Expression Native hIL-13 Construct Backmutations μg/ml/ STAT6assay VH VL VH COS; 48 h IC 50, nM V2.0 V2 None, CDR grafted 8-10 >100CDR graft V 2.1 V2 V48I; A49G  9-14 2.8 V 2.2 V2 R67K; F68A 5-6 >100 V2.3 V2 R72A 8-9 1.67-2.6 V 2.4 V2 A49G 10 17.5 V 2.5 V2 R67K; F68A; R72A4-5 1.75 V 2.6 V2 V48I; A49G: R72A 11-12 1.074-3.37 V 2.7 V2 A49G; R72A10-11 1.7 V 2.11 V2 V48I; A49G: 24 0.25-0.55 R72A: L79A

Example 12 Binding Interaction Site Between IL-13 and IL-13RI1

A complex of IL-13, the extracellular domain of IL-13RI1 (residues27-342 of SEQ ID NO:125), and an antibody that binds human IL-13 wasstudied by x-ray crystallography. See, e.g., 16163-029001. Two points ofsubstantial interaction were found between IL-13 and IL-13Rα1. Theinteraction between Ig domain 1 of IL-13Rα1 and IL-13 results in theformation of an extended beta sheet spanning the two molecules. ResiduesThr88 [Thr107], Lys89 [Lys108], Ile90 [Ile109], and Glu91 [Glu110] ofIL-13 (SEQ ID NO:124, mature sequence [full-length sequence (SEQ IDNO:178)]) form a beta strand that interacts with residues Lys76, Lys77,Ile78 and A1a79 of the receptor (SEQ ID NO:125). Additionally, the sidechain of Met33 [Met52] of IL-13 (SEQ ID NO:124 [SEQ ID NO:178]) extendsinto a hydrophobic pocket that is created by the side chains of theseadjoining strands.

The predominant feature of the interaction with Ig domain 3 is theinsertion of a hydrophobic residue (Phe107 [Phe126]) of IL-13 (SEQ IDNO:124 [SEQ ID NO:178]) into a hydrophobic pocket in Ig domain 3 of thereceptor IL-13Rα1. The hydrophobic pocket of IL-13Rα1 is formed by theside chains of residues Leu319, Cys257, Arg256, and Cys320 (SEQ IDNO:125). The interaction with Phe107 [Phe126] of IL-13 (SEQ ID NO:124[SEQ ID NO:178]) results in an extensive set of van der Waalsinteractions between amino acid residues Ile254, Ser255, Arg256, Lys318,Cys320, and Tyr321 of IL-13Rα1 (SEQ ID NO:125) and amino acid residuesArg11 [Arg30], Glu12 [Glu31], Leu13 [Leu32], Ile14 [Ile33], Glu15[Ile34], Lys104 [Lys123], Lys105 [Lys124], Leu106 [Leu125], Phe107[Phe126], and Arg108 [Arg 127] of IL-13 (SEQ ID NO:124 [SEQ ID NO:178]).These results demonstrate that an IL-13 binding agent that binds to theregions of IL-13 involved in interaction with IL-13RI1 can be used toinhibit IL-13 signaling.

Example 13 Expression of Humanized MJ 2-7 Antibody in COS Cells

To evaluate the production of chimeric anti-NHP IL13 antibodies in themammalian recombinant system, the variable regions of mouse MJ 2-7antibody were subcloned into a pED6 expression vector containing humankappa and IgG1mut constant regions. Monkey kidney COS-1 cells were grownin DME media (Gibco) containing 10% heat-inactivated fetal bovine serum,1 mM glutamine and 0.1 mg/ml Penicillin/Streptomycin. Transfection ofCOS cells was performed using TRANSITIT3-LT1 Transfection reagent(Mirus) according to the protocol suggested by the reagent supplier.Transfected COS cells were incubated for 24 hours at 37° C. in thepresence of 10% CO₂, washed with sterile PBS, and then grown inserum-free media R1CD1 (Gibco) for 48 hours to allow antibody secretionand accumulation in the conditioned media. The expression of chMJ 2-7antibody was quantified by total human IgG ELISA using purified humanIgG1/kappa antibody as a standard.

The production of chimeric MJ 2-7 antibody in COS cells wassignificantly lower then the control chimeric antibody (Table 2).Therefore, optimization of Ab expression was included in the MJ 2-7humanization process. The humanized MJ 2-7 V1 was constructed by CDRgrafting of mouse MJ 2-7 heavy chain CDRs onto the most homologous humangermline clone, DP 25, which is well expressed and represented intypical human antibody response. The CDRs of light chain were subclonedonto human germline clone DPK 18 in order to generate huMJ 2-7 V1 VL.The humanized MJ 2-7 V2 was made by CDR grafting of CDRs MJ 2-7 heavychain variable region onto DP54 human germline gene framework and CDRsof MJ 2-7 light chain variable region onto DPK9 human germline geneframework. The DP 54 clone belongs to human VH III germline subgroup andDPK9 is from the V kappa I subgroup of human germline genes. Antibodymolecules that include VH III and V kappa I frameworks have highexpression level in E. coli system and possess high stability andsolubility in aqueous solutions (see, e.g., Stefan Ewert et al., J. Mol.Biol. (2003), 325; 531-553, Adrian Auf et al., Methods (2004)34:215-224). We have used the combination of DP54/DPK9 human frameworksin the production of several recombinant antibodies and have achieved ahigh expression of antibody (>20 Tg/ml) in the transient COStransfection experiments.

TABLE 2 mAb Expression, Tg/ml 3D6 10.166 Ch MJ 2-7 pED6 (1) 2.44 Ch MJ2-7 pED6 (2) 2.035 h12A11 V2 1.639

The CDR grafted MJ 2-7 V1 and V2 VH and VL genes were subcloned into twomammalian expression vector systems (pED6kappa/pED6 IgG1mut andpSMEN2kappa/pSMED2IgG1mut), and the production of humanized MJ 2-7antibodies was evaluated in transient COS transfection experiments asdescribed above. In the first set of the experiments the effect ofvarious combinations of huMJ 2-7 VL and VH on the antibody expressionwas evaluated (Table 3). Changing of MJ 2-7 VL framework regions to DKP9increased the antibody production 8-10 fold, whereas VL V1 (CDR graftedonto DPK 18) showed only a moderate increase in antibody production.This effect was observed when humanized VL was combined with chimeric MJ2-7 VH and humanized MJ 2-7 V1 and V2. The CDR grafted MJ 2-7 V2 had a3-fold higher expression level then CDR grafted MJ 2-7 V1 in the sameassay conditions.

TABLE 3 mAb Expression, Tg/ml ChMJ 2-7 1.83 hVH V1/mVL 3.04 hVH V1/hVLV1 6.34 hVH V1/hVL V2 15.4 hVH-V2/mVL 0.2 mVH/hVL-V2 18.41 hVH-V2/hVL-V15.13 hVH-V2/hVL-V2 10.79

Similar experiments were performed with huMJ 2-7 V2 containing backmutations in the heavy chain variable regions (Table 4). The highestexpression level was detected for huMJ 2-7 V2.11 that retained theantigen binding and neutralization properties of mouse MJ 2-7 antibody.Introduction of back mutations at the positions 48 and 49 (V48I andA49G) increased the production of huMJ 2-7 V2 antibody in COS cells,whereas the back mutations of amino acids at the positions 23, 24, 67and 68 (A23T; A24G; R67K and F68A) had a negative impact on antibodyexpression.

TABLE 4 mAb Expression, Tg/ml V2 8.27 V2.1 12.1 V2.2 5.29 V2.3 9.60 V2.48.20 V2.5 6.05 V2.6 11.3 V2.10 9.84 V2.11 14.85 V2.16 1.765

Example 14 Evaluation of Antigen Binding Properties of Humanized MJ 2-7Antibodies by NHP IL-13 FLAG ELISA

The ability of fully humanized MJ 2-7 mAb (V1, V2 v2) to compete withbiotinylated mouse MJ 2-7 Ab for binding to NHP IL-13-FLAG was evaluatedby ELISA. The microtiter plates (Costar) were coated with 1 μg/ml ofanti-FLAG monoclonal antibody M2 (Sigma). The FLAG NHP IL-13 protein atconcentration of 10 ng/ml was mixed with 10 ng/ml of biotin labeledmouse MJ 2-7 antibody and various concentrations of unlabeled mouse andhumanized MJ 2-7 antibody. The mixture was incubated for 2 hours at roomtemperature and then added to the anti-FLAG antibody-coated plate.Binding of FLAG NHP-IL-13/bioMJ2-7 Ab complexes was detected withstreptavidin-HRP and 3,3′,5,5′-tetramethylbenzidine (TMB). The humanizedMJ 2-7 V2 significantly lost activity whereas huMJ 2-7 V2.11 completelyrestored the antigen binding activity and was capable of competing withbiotinylated MJ 2-7 mAb for binding to FLAG-NHP IL-13. BIACORE™ analysisalso confirmed that NHP IL-13 had rapid binding to and slow dissociationto immobilized hluMJ 2-7 v2.11.

Example 15 Molecular Modeling of Humanized MJ2-7 V2VH

Structure templates for modeling humanized MJ2-7 heavy chain version 2(MJ2-7 V2VH) were selected based on BLAST homology searches againstProtein Data Bank (PDB). Besides the two structures selected from theBLAST search output, an additional template was selected from anin-house database of protein structures. Model of MJ2-7 V2VH was builtusing the three template structures 1JPS (co-crystal structure of humantissue factor in complex with humanized Fab D3h44), 1N8Z (co-crystalstructure of human Her2 in complex with Herceptin Fab) and F13.2 (IL-13in complex with mouse antibody Fab fragment) as templates and theHomology module of InsightII (Accelrys, San Diego). The structurallyconserved regions (SCRs) of 1JPS, 1N8Z and F13.2 (available from16163-029001) were determined based on the Cα distance matrix for eachmolecule and the template structures were superimposed based on minimumRMS deviation of corresponding atoms in SCRs. The sequence of the targetprotein MJ2-7 V2VH was aligned to the sequences of the superimposedtemplates proteins and coordinates of the SCRs were assigned to thecorresponding residues of the target protein. Based on the degree ofsequence similarity between the target and the templates in each of theSCRs, coordinates from different templates were used for different SCRs.Coordinates for loops and variable regions not included in the SCRs weregenerated by Search Loop or Generate Loop methods as implemented inHomology module. Briefly, Search Loop method scans protein structuresthat would fit properly between two SCRs by comparing the Ca distancematrix of flanking SCR residues with a pre-calculated matrix derivedfrom protein structures that have the same number of flanking residuesand an intervening peptide segment of a given length. Generate Loopmethod that generate atom coordinates de novo was used in those caseswhere Search Loops did not produce desired results. Conformation ofamino acid side chains was kept the same as that in the template if theamino acid residue was identical in the template and the target.However, a conformational search of rotamers was done and theenergetically most favorable conformation was retained for thoseresidues that are not identical in the template and target. This wasfollowed by Splice Repair that sets up a molecular mechanics simulationto derive proper bond lengths and bond angles at junctions between twoSCRs or between SCR and a variable region. Finally the model wassubjected to energy minimization using Steepest Descents algorithm untila maximum derivative of 5 kcal/(mol Å) or 500 cycles and ConjugateGradients algorithm until a maximum derivative of 5 kcal/(mol Å) or 2000cycles. Quality of the model was evaluated using ProStat/Struct_Checkcommand.

Molecular model of mouse MJ2-7 VH was built by following the proceduredescribed for humanized MJ2-7 V2VH except the templates used were 1QBLand 1QBM, crystal structures for horse anti-cytochrome c antibody FabE8.

Potential differences in CDR-Framework H-bonds predicted by the models

hMJ2-7 V2VH:G26-hMJ2-7 V2VH:A24

hMJ2-7 V2VH:Y109-hMJ2-7 V2VH:S25

mMJ2-7 VH:D61-mMJ2-7 VH:148

mMJ2-7 VH:K63-mMJ2-7 VH:E46

mMJ2-7 VH:Y109-mMJ2-7 VH:R98

These differences suggested the following optional back mutations: A23T,A24G and V481.

Other optional back mutations suggested based on significant RMSdeviation of individual amino acids and differences in amino acidresidues adjacent to these are: G9A, L115T and R87T.

Example 16 IL-13 Neutralization Activity of MJ2-7 and C65

The IL-13 neutralization capacities of MJ2-7 and C65 were tested in aseries of bioassays. First, the ability of these antibodies toneutralize the bioactivity of NHP IL-13 was tested in a monocyte CD23expression assay. Freshly isolated human PBMC were incubated overnightwith 3 ng/ml NHP IL-13 in the presence of increasing concentrations ofMJ2-7, C65, or sIL-13RI2-Fc. Cells were harvested, stained withCYCHROME3-labeled antibody to the monocyte-specific marker, CD11b, andwith PE-labeled antibody to CD23. In response to IL-13 treatment, CD23expression is up-regulated on the surface of monocytes, which were gatedbased on expression of CD11b. MJ2-7, C65, and sIL13RI2-Fc all were ableto neutralize the acitivity of NHP IL-13 in this assay. The potencies ofMJ2-7 and sIL-13RI2-Fc were equivalent. C65 was approximately 20-foldless active (FIG. 2).

In a second bioassay, the neutralization capacities of MJ2-7 and C65 fornative human IL-13 were tested in a STAT6 phosphorylation assay. TheHT-29 epithelial cell line was incubated with 0.3 ng/ml native humanIL-13 in the presence of increasing concentrations of MJ2-7, C65, orsIL-13RI2-Fc, for 30 minutes at 37° C. Cells were fixed, permeabilized,and stained with ALEXA3 Fluor 488-labeled antibody to phosphorylatedSTAT6. IL-13 treatment stimulated STAT6 phosphorylation. MJ2-7, C65, andsIL13Ra2-Fc all were able to neutralize the acitivity of native humanIL-13 in this assay (FIG. 3). The IC50's for the murine MJ-27 antibodyand the humanized form (V2.11) were 0.48 nM and 0.52 nM respectively.The potencies of MJ2-7 and sIL-13RI2-Fc were approximately equivalent.The IC50 for sIL-13Ra2-Fc was 0.33 nM (FIG. 4). C65 was approximately20-fold less active (FIG. 5).

In a third bioassay, the ability of MJ2-7 to neutralize native humanIL-13 was tested in a tenascin production assay. The human BEAS-2B lungepithelial cell line was incubated overnight with 3 ng/ml native humanIL-13 in the presence of increasing concentrations of MJ2-7.Supernatants were harvested and tested for production of theextracellular matrix protein, tenascin C, by ELISA (FIG. 6A). MJ2-7inhibited this response with IC50 of approximately 0.1 nM (FIG. 6B).

These results demonstrate that MJ2-7 is an effective neutralizer of bothNHP IL-13 and native human IL-13. The IL-13 neutralization capacity ofMJ2-7 is equivalent to that of sIL-13RI2-Fc. MJ1-65 also has IL-13neutralization activity, but is approximately 20-fold less potent thanMJ2-7.

Example 17 Epitope Mapping of MJ2-7Antibody by SPR

sIL-13RI2-Fc was directly coated onto a CM5 chip by standard aminecoupling. NHP-IL-13 at 100 nM concentration was injected, and itsbinding to the immobilized IL-13RI2-Fc was detected by BIACORE3. Anadditional injection of 100 nM of anti IL-13 antibodies was added, andchanges in binding were monitored. MJ2-7 antibody did not bind toNHP-IL-13 when it was in a complex with hu IL-13RI2, whereas a positivecontrol anti-IL-13 antibody did (FIG. 7). These results indicate that huIL-13RI2 and MJ2-7 bind to the same or overlapping epitopes of NHPIL-13.

Example 18 Measurement of Kinetic Rate Constants for the InteractionBetween NHP-IL-13 and Humanized MJ2-7 V2-11 Antibody

To prepare the biosensor surface, goat anti-human IgG Fc specificantibody was immobilized onto a research-grade carboxy methyl dextranchip (CM5) using amine coupling. The surface was activated with amixture of 0.1 M1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and0.05 M N-Hydroxysuccinimide (NHS). The capturing antibody was injectedat a concentration of 10 Tg/ml in sodium acetate buffer (pH 5.5).Remaining activated groups were blocked with 1.0 M ethanolamine (pH8.0). As a control, the first flow cell was used as a reference surfaceto correct for bulk refractive index, matrix effects, and non-specificbinding, the second, third and fourth flow cells were coated with thecapturing molecule.

For kinetic analysis, the monoclonal antibody hMJ2-7 V2-11 was capturedonto the anti IgG antibody surface by injecting 40 Tl of a 1 Tg/mlsolution. The net difference between the baseline and the pointapproximately 30 seconds after completion of injection was taken torepresent the amount of target bound. Solutions of NHP-IL-13 at 600,200, 66.6, 22.2, 7.4, 2.5, 0.8, 0.27, 0.09 and 0 nM concentrations wereinjected in triplicate at a flow rate of 100 Tl per min for 2 minutes,and the amount of bound material as a function of time was recorded(FIG. 8). The dissociation phase was monitored in HBS/EP buffer (10 mMHEPES, pH 7.4, containing 150 mM NaCl, 3 mM EDTA and 0.005% (v/v)Surfactant P20) for 5 minutes at the same flow rate followed by two 5 Tlinjections of glycine, pH 1.5, to regenerate a fully active capturingsurface. All kinetic experiments were done at 22.5° C. in HBS/EP buffer.Blank and buffer effects were subtracted for each sensorgram usingdouble referencing.

The kinetic data were analyzed using BIAEVALUATION3 software 3.0.2applied to a 1:1 model. The apparent dissociation (kd) and association(ka) rate constants were calculated from the appropriate regions of thesensorgrams using a global analysis. The affinity constant of theinteraction between antibody and NHP IL-13 was calculated from thekinetic rate constants by the following formula: Kd=kd/ka. These resultsindicate that huMJ2-7 V2-11 has on and off-rates of 2.05×10⁷ M⁻¹s⁻¹ and8.89×10⁻⁴ l/s, respectively, resulting in an antibody with 43 μMaffinity for NHP-IL-13.

Example 19 Inhibitory Activity of MJ2-7 Humanization Intermediates inBioassays

The inhibitory activity of various intermediates in the humanizationprocess was tested by STATE phosphorylation and tenascin productionbioassays. A sub-maximal level of NHP IL-13 or native human IL-13 crudepreparation was used to elicit the biological response, and theconcentration of the humanized version of MJ2-7 required forhalf-maximal inhibition of the response was determined. Analysis hMJ2-7V1, hMJ2-7 V2 and hMJ2-7 V3, expressed with the human IgG1, and kappaconstant regions, showed that Version 2 retained neutralization activityagainst native human IL-13. This concentration of the Version 2humanized antibody required for half-maximal inhibition of native humanIL-13 bioactivity was approximately 110-fold greater than that of murineMJ2-7 (FIG. 9). Analysis of a semi-humanized form, in which the V1 or V2VL was combined with murine MJ2-7 VH, demonstrated that the reduction ofnative human IL-13 neutralization activity was not due to to thehumanized VL, but rather to the VH sequence (FIG. 10). Whereas thesemi-humanized MJ2-7 antibody with VL V1 only partially retained theneutralization activity the version with humanized VL V2 was as activeas parental mouse antibody. Therefore, a series of back-mutations wereintroduced into the V1 VH sequence to improve the native human IL-13neutralization activity of murine MJ2-7.

Example 20 MJ2-7 Blocks IL-13 Interaction with IL-13RI1 and IL-13RI2

MJ2-7 is specific for the C-terminal 19-mer of NHP IL-13, correspondingto amino acid residues 114-132 of the immature protein (SEQ ID NO:24),and residues 95-113 of the mature protein (SEQ ID NO:14). For humanIL-13, this region, which forms part of the D alpha-helix of theprotein, has been reported to contain residues important for binding toboth IL-13RI1 and IL-13RI2. Analysis of human IL-13 mutants identifiedthe A, C, and D-helices as containing important contacts site for theIL-13RI1/IL-4RI signaling complex (Thompson and Debinski (1999) J. Biol.Chem. 274: 29944-50). Alanine scanning mutagenesis of the D-helixidentified residues K123, K124, and R127 (SEQ ID NO:24) as responsiblefor interaction with IL-13RI2, and residues E110, E128, and L122 asimportant contacts for IL-13RI1 (Madhankmuar et al. (2002) J. Biol.Chem. 277: 43194-205). High resolution solution structures of humanIL-13 determined by NMR have predicted the IL-13 binding interactionsbased on similarities to related ligand-receptor pairs of knownstructure. These NMR studies have supported a key role for the IL-13 Aand D-helices in making important contacts with IL-13RI1 (Eisenmesser etal. (2001) J. Mol. Biol. 310:231-241; Moy et al. (2001) J. Mol. Biol.310:219-230). Binding of MJ2-7 to this epitope located in theC-terminal, D-helix of IL-13 was predicted to disrupt interaction ofIL-13 with IL-13RI1 and IL-13RI2.

The ability of MJ2-7 to inhibit binding of NHP IL-13 to IL-13RI1 andIL-13RI2 was tested by ELISA. Recombinant soluble forms of humanIL-13RI1-Fc and IL-13RI2-Fc were coated onto ELISA plates. FLAG-taggedNHP IL-13 was added in the presence of increasing concentrations ofMJ2-7. Results showed that MJ2-7 competed with both soluble receptorforms for binding to NHP IL-13 (FIGS. 11A and 11B). This provides abasis for the neutralization of IL-13 bioactivity by MJ2-7.

Example 21 The MJ 2-7 Light Chain CDRs Contribute to Antigen Binding

To evaluate if all three light chain CDR regions are required for thebinding of MJ 2-7 antibody to NHP IL-13, two additional humanizedversions of MJ 2-7 VL were constructed by CDR grafting. The VL version 3was designed based on human germline clone DPK18, contained CDR1 andCDR2 of the human germline clone and CDR3 from mouse MJ2-7 antibody(FIG. 12). In the second construct (hMJ 2-7 V4), only CDR1 and CDR2 ofMJ 2-7 antibody were grafted onto DPK 18 framework, and CDR3 was derivedfrom irrelevant mouse monoclonal antibody.

The humanized MJ 2-7 V3 and V4 were produced in COS cells by combininghMJ 2-7 VH V1 with hMJ 2-7 VL V3 and V4. The antigen binding propertiesof the antibodies were examined by direct NHP IL-13 binding ELISA. ThehMJ 2-7 V4 in which MJ 2-7 light chain CDR3 was absent retained theability to bind NHP IL-13, whereas V3 that contained human germline CDR1and CDR2 in the light chain did not bind to immobilized NHP IL-13. Theseresults demonstrate that CDR1 and CDR2 of MJ 2-7 antibody light chainare most likely responsible for the antigen binding properties of thisantibody.

Nucleotide sequence of hMJ 2-7 VL V3 (SEQ ID NO: 189) 1ATGCGGCTGC CCGCTCAGCT GCTGGGCCTG CTGATGCTGT GGGTGCCCGG 51CTCTTCCGGC GACGTGGTGA TGACCCAGTC CCCTCTGTCT CTGCCCGTGA 101CCCTGGGCCA GCCCGCTTCT ATCTCTTGCC GGTCCTCCCA GTCCCTGGTG 151TACTCCGACG GCAACACCTA CCTGAACTGG TTCCAGCAGA GACCCGGCCA 201GTCTCCTCGG CGGCTGATCT ACAAGGTGTC CAACCGCTTT TCCGGCGTGC 251CCGATCGGTT CTCCGGCTCC GGCAGCGGCA CCGATTTCAC CCTGAAGATC 301AGCCGCGTGG AGGCCGAGGA TGTGGGCGTG TACTACTGCT TCCAGGGCTC 351CCACATCCCT TACACCTTTG GCGGCGGAAC CAAGGTGGAG ATCAAGAmino acid sequence of hMJ 2-7 VL V3 (SEQ ID NO: 190)MRLPAQLLGLLMLWVPGSSG-DVVMTQSPLSLPVTLGQPASISC RSSQ SLVYSDGNTYLNWFQQRPGQSPRRLIY KVSNRFS GVPDRFSGSGSGTD FTLKISRVEAEDVGVYYC FQGSHIPYTFGGGTKVEIK Nucleotide sequence of hMJ 2-7 VL V4 (SEQ ID NO: 191)GATGTTGTGATGACCCAATCTCCACTCTCCCTGCCTGTCACTCCTGGAGAGCCAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTGCATAGTAATGGAAACACCTACCTGGAATGGTACCTGCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCTTTCAAAGTTCACATGTTCCTCTCACCTTCGGTCAGGGGACCAAGCTGGAGATCAAAAmino acid sequence of hMJ 2-7 VL V4 (SEQ ID NO: 192)DVVMTQSPLS LPVTPGEPAS ISC RSSQSIV   HSNGNTYLE W YLQKPGQSPQ LLIY KVSNRF  S GVPDRFSGS GSGTDFTLKISRVEA EDVGV YYC FQSSHVP LT FGQGTKLE IK

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments described herein described herein. Other embodiments arewithin the following claims.

1-35. (canceled)
 36. A method of treating an immune disorder in patientcomprising administering to the patient an effective amount of an IL-13antibody comprising a heavy chain immunoglobulin variable domainsequence and a light chain immunoglobulin variable domain that binds toIL-13 with a K_(D) of less than 10⁻⁷ M, wherein the heavy chain variabledomain comprises the amino acid sequence of: (SEQ ID NO: 48) (i)G-(YF)-(NT)-I-K-D-T-Y-(MI)-H in CDR1, (SEQ ID NO: 49) (ii)(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-G in CDR2, and(SEQ ID NO: 17) (iii) SEENWYDFFDY in CDR3;

and wherein the light chain variable domain comprises the amino acidsequence of: (SEQ ID NO: 25) (i)(RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y- L-(EDNQYAS) in CDR1,(SEQ ID NO: 27) (ii) K-(LVI)-S-(NY)-(RW)-(FD)-S in CDR2, and(SEQ ID NO: 28) (iii) Q-(GSA)-(ST)-(HEQ)-I-P in CDR3.


37. The method of claim 36, wherein the heavy chain variable domaincomprises: GFNIKDTYIH (SEQ ID NO:15), in CDR1, RIDPANDNIKYDPKFQG (SEQ IDNO:16), in CDR2, and SEENWYDFFDY (SEQ ID NO:17), in CDR3; and whereinthe light chain variable domain sequence comprises: RSSQSIVHSNGNTYLE(SEQ ID NO:18), in CDR1, KVSNRFS (SEQ ID NO:19), in CDR2, and FQGSHIPYT(SEQ ID NO:20), in CDR3.
 38. The method of claim 36, wherein theantibody is a recombinant IgG that includes an Fc domain.
 39. The methodof claim 36, wherein the antibody comprises an Fc domain that is mutatedto reduce on or more of Fc receptor binding, antibody glycosylation,number of cysteine residues, effector cell function or complementfunction.
 40. The method of claim 36, wherein the antibody is a Fab orscFv.
 41. The method of claim 36, wherein the antibody comprises humanframework regions, a human Fc region, or both.
 42. The method of claim36, wherein the frameworks of the heavy chain domain sequence comprise:(i) at a position corresponding to 49, Gly; (ii) at a positioncorresponding to 72, Ala; (iii) at a position corresponding to 48, Ile,and to 49, Gly; (iv) at a position corresponding to 48, Ile, to 49, Gly,and to 72, Ala; (v) at a position corresponding to 67, Lys, to 68, Ala,and to 72, Ala; and/or (vi) at a position corresponding to 48, Ile, to49, Gly, to 72, Ala, to 79, Ala.
 43. The method of claim 36, wherein theheavy chain variable domain sequence comprises the amino acid sequenceof one or more of: GFNIKDTYIH (SEQ ID NO:15), in CDR1, R1DPANDNIKYDPKFQG(SEQ ID NO:16), in CDR2, or SEENWYDFFDY (SEQ ID NO:17), in CDR3.
 44. Themethod of claim 36, wherein the light chain variable domain sequencecomprises the amino acid sequence of one more more of: RSSQSIVHSNGNTYLE(SEQ ID NO:18), in CDR1, KVSNRFS (SEQ ID NO:19), in CDR2, or FQGSHIPYT(SEQ ID NO:20), in CDR3.
 45. The method of claim 36, wherein theantibody molecule is an isolated, recombinant IgG antibody thatcomprises two polypeptide chains: a light chain that comprises the lightchain variable domain of V2.11 (SEQ ID NO:36) and a heavy chain thatcomprises the heavy chain variable domain of V2.1 (SEQ ID NO:71), V2.3(SEQ ID NO:73), V2.4 (SEQ ID NO:74), V2.5 (SEQ ID NO:75), V2.6 (SEQ IDNO:76), V2.7 (SEQ ID NO:77), or V2.11 (SEQ ID NO:80).
 46. The method ofclaim 36, wherein the immune disorder is an IL-13 related disorder. 47.The method of claim 36, wherein the immune disorder is selected from thegroup consisting of: asthmatic disorders, atopic disorders, chronicobstructive pulmonary disease, conditions involving airway inflammation,eosinophilia, fibrosis and excess mucus production, inflammatoryconditions, autoimmune conditions, tumors or cancers, viral infection,inflammatory bowel disease, Crohn's disease, and ulcerative colitis. 48.The method of claim 47, wherein the immune disorder is ulcerativecolitis.